CN104919055B - With the method and composition of single signal detection multiple analytes - Google Patents

With the method and composition of single signal detection multiple analytes Download PDF

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Publication number
CN104919055B
CN104919055B CN201380022600.1A CN201380022600A CN104919055B CN 104919055 B CN104919055 B CN 104919055B CN 201380022600 A CN201380022600 A CN 201380022600A CN 104919055 B CN104919055 B CN 104919055B
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analyte
unit
interest
interaction
bridge
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CN104919055A (en
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尼古拉斯·西斯利亚诺
路易斯·莱昂
马丁·帕特里克·基奥
阿什利·沙尼斯·布朗
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Invisible Sentinel Inc
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Invisible Sentinel Inc
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54306—Solid-phase reaction mechanisms
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5023—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures with a sample being transported to, and subsequently stored in an absorbent for analysis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6804—Nucleic acid analysis using immunogens
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0627—Sensor or part of a sensor is integrated
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/069—Absorbents; Gels to retain a fluid
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
    • G01N33/553—Metal or metal coated

Abstract

Offer detects the composition of multiple analytes, method and apparatus with single signal.

Description

With the method and composition of single signal detection multiple analytes
Technical field
Embodiment detects multiple analytes directed in part to single signal.
Background technology
Detection multiple analytes usually require using multiple signals or multiple reactions, point or hole to determine whether sample has Multiple analytes.This can make explanation become complicated, and be classified as can detect with two or more in dopant In the case of feature, identification can be made challenging for end user.Therefore, in order to simplify and end user is carried For comprehensive qualitative report, it would be desirable to be able to the method and composition of the multiple analytes in enough single signal detection samples.This hair It is bright to meet this needs and other needs.
Invention content
The present invention provides the side for concurrently (simultaneously, concurrently) detecting the first analyte and the second analyte Method, including：Make solid support and the first analyte, the second analyte, the bridge-jointing unit comprising the second capture agent and comprising The detecting signal unit contact of third capture agent；With the existence or non-existence of detection detecting signal unit, the signal detection Unit concurrently indicates the existence or non-existence of the first analyte and the second analyte, wherein the first capture agent fix (adherency, It is attached, affix) in solid support；First analyte include be incorporated into the first capture agent first interaction unit and It is incorporated into the second interaction unit of bridge-jointing unit；And second the first interaction for including with reference to bridge-jointing unit of analyte it is single Member and the second interaction unit for being incorporated into detecting signal unit.
The present invention also provides the sides that the first analyte, the second analyte and third analyte are concurrently detected with single signal Method, including：First, second, and third analyte is made to be examined with solid support, the first bridge-jointing unit, the second bridge-jointing unit and signal Survey unit contact；With the presence of detection detecting signal unit, detecting signal unit concurrently indicates first, second with single signal With the presence of third analyte, wherein：First analyte includes the first interaction unit and the second interaction unit；Second Analyte includes the first interaction unit and the second interaction unit；Third analyte include first interaction unit and Second interaction unit；Solid support includes the first capture examination of the first interaction unit for being incorporated into the first analyte Agent；First bridge-jointing unit is incorporated into the second interaction unit of the first analyte and the first interaction list of the second analyte Member；Second bridge-jointing unit is incorporated into the second interaction unit of the second analyte and the first interaction list of third analyte Member；And detecting signal unit is incorporated into the second interaction unit of third analyte.Unit interact in each analyte On can be different from each other.
In some embodiments, the method for concurrently detecting the first analyte and the second analyte is provided, method includes： Make solid support and the first analyte of interest, the second analyte of interest, the bridge joint list comprising the second capture agent Member and the detecting signal unit contact comprising third capture agent；It is described with the existence or non-existence of detection detecting signal unit Detecting signal unit concurrently indicates the existence or non-existence of the first analyte of interest and the second analyte of interest, In：First capture agent is fixed on solid support；First analyte of interest, which includes, is incorporated into the of the first capture agent One interaction unit and the second interaction unit for being incorporated into bridge-jointing unit；And the second analyte of interest includes knot Close the first interaction unit of bridge-jointing unit；Be incorporated into the second analyte, the second analyte first interaction unit or Second interaction unit, the first and second analytes compound or only deposited when compound contains the first and second analytes Bridge-jointing unit component detecting signal unit.
Embodiments described herein also provides single comprising solid support, the first analyte, the second analyte, bridge joint The compound of member and detecting signal unit, each member of wherein compound are directly or indirectly bonded to each other.
Embodiments described herein is also provided comprising solid support, the first analyte, the second analyte, third point Analyse the compound of object, the first bridge-jointing unit, the second bridge-jointing unit and detecting signal unit, wherein solid support, the first analysis Object, the second analyte, third analyte, the first bridge-jointing unit, the second bridge-jointing unit and detecting signal unit are directly or indirectly It is bonded to each other.
Provided herein is the methods for concurrently detecting the first analyte of interest and the second analyte of interest.At some In embodiment, method includes making solid support and the first analyte of interest, the second analyte of interest, comprising the The bridge-jointing unit of two capture agents and the detecting signal unit contact comprising third capture agent；With detection detecting signal unit Existence or non-existence, the detecting signal unit concurrently indicate the first analyte of interest and the second analyte of interest Existence or non-existence, wherein the first capture agent is fixed on solid support；First analyte of interest is included and is incorporated into First interaction unit of the first capture agent and the second interaction unit for being incorporated into bridge-jointing unit；It is and of interest Second analyte includes the first interaction unit and the second interaction unit, wherein the first interaction unit combines bridge joint Unit；It is incorporated into following detecting signal unit：I) the second analyte, ii) the second analyte first interaction unit or Second interaction unit, iii) the first and second analytes compound component or iv) only when compound contains the first He The component of existing analyte-bridge joint compound during the second analyte.
In some embodiments, the first and second interaction units and of interest of the first analyte of interest The second analyte first and second interaction units be each independently heterologous interaction unit.In some embodiment party In formula, the second interaction unit of the first analyte of interest and the first interaction of the second analyte of interest are single Member includes identical heterologous interaction unit.In some embodiments, the second phase interaction of the first analyte of interest Different heterologous interaction units is included with the first interaction unit of unit and the second analyte of interest.At some In embodiment, the first interaction unit of the first analyte of interest and the second of the second analyte of interest is mutually Action cell includes identical heterologous interaction unit.In some embodiments, the first of the first analyte of interest Interaction unit and the second interaction unit of the second analyte of interest include different heterologous interaction units.
Offer concurrently detects the first analyte of interest, the second analyte of interest and of interest with single signal Third analyte method.In some embodiments, method include make first, second, and third analyte of interest with Solid support, the first bridge-jointing unit, the second bridge-jointing unit and detecting signal unit contact；With depositing for detection detecting signal unit , the detecting signal unit concurrently indicates the presence of first, second, and third analyte of interest with single signal, In：First analyte of interest includes the first interaction unit and the second interaction unit；Second analysis of interest Object includes the first interaction unit and the second interaction unit；It is single that third analyte of interest includes the first interaction Member and the 5th interaction unit；Solid support includes the first interaction unit for being incorporated into the first analyte of interest The first capture agent；First bridge-jointing unit is incorporated into the second interaction unit and of interest of the first analyte of interest The second analyte first interaction unit；Second bridge-jointing unit be incorporated into the second analyte of interest second mutually First interaction unit of action cell and third analyte of interest；And detecting signal unit is incorporated into：I) third Analyte, ii) third analyte first interaction unit or second interaction unit, iii) first, second or third The component or iv of analyte complex) existing analyte-bridge joint only when compound contains first, second, and third analyte The component of compound.
In some embodiments, bridge-jointing unit described herein is multivalence capture agent.In some embodiments, Multivalence capture agent is immunoglobulin.In some embodiments, immunoglobulin IgM.Bridge-jointing unit can also make a living Object element.
The method that use device concurrently detects multiple analytes with single signal is provided.In some embodiments, side Method includes a) contacting with one or more samples comprising multiple analytes using the device of single signal detection multiple analytes, Wherein device includes：Shell, including：The import contacted with bonding pad (conjugate pad, conjugate pad) in fluid (inlet opening)；Force member (force member)；Contact the slidably locking component (slidable of force member locking member)；Contact the connecting elements (attachment member) of force member；Connect the slip of component Button (sliding button)；With include bonding pad, test film (test membrane) and absorption component (absorbent Member detection membranous system (detection membrane system)), bonding pad, test film and absorption component are at least A part is substantially parallel to each other, and force member contact detects membranous system and can apply substantially perpendicular to detection membranous system Pressure, sliding button movement slidably locking component, bonding pad includes the detecting signal unit for including third capture agent；It surveys Examination film includes the first capture agent for being fixed on test film；Wherein one or more samples include the first analyte of interest, Second analyte of interest and the bridge-jointing unit for including the second capture agent, combine wherein the first analyte of interest includes In the first capture agent the first interaction unit and be incorporated into the second interaction unit of bridge-jointing unit, it is and of interest The second analyte include with reference to bridge-jointing unit first interaction unit and second interaction unit；Wherein include third The detecting signal unit of capture agent is incorporated into：I) the second analyte, ii) the second analyte the first interaction unit or the Two interaction units, iii) the first analyte and the second analyte complex component or iv) only when compound contains first With the component of analyte-bridge joint compound existing during the second analyte；And it b) detects the presence of detecting signal unit or does not deposit The presence or not of the first analyte of interest and the second analyte of interest is concurrently indicated in, the detecting signal unit In the presence of.
In some embodiments, method is included in the parts of one or more samples and has contacted and flowed through combination Bonding pad is moved after pad, thus makes at least part exposure of test film for detecting detecting signal unit, so as to use Single signal indicates the existence or non-existence of multiple analytes.In some embodiments, bonding pad is by making slidably to lock Determine component movement to move.In some embodiments, the first analyte and the second analyte are amplicon.In some implementations In mode, the first analyte and the second analyte are PCR reaction products.In some embodiments, the first of the first analyte Interaction unit marks (label, label) for digoxin (digoxigenin).In some embodiments, the first analyte Second interaction unit for rhodamine (rhodamine) label.In some embodiments, the first phase of the second analyte Interaction unit is marked for rhodamine.In some embodiments, the second interaction unit of the second analyte is fluorescein Label.In some embodiments, third capture agent is incorporated into the second interaction unit of the second analyte.In some realities It applies in mode, third capture agent is biotinylated capture agent.In some embodiments, signal interaction unit applies It is furnished with Streptavidin (streptavidin).In some embodiments, signal interaction unit is applied for Streptavidin The colloidal gold (aurosol) of cloth.In some embodiments, the first and second analytes are nucleic acid amplification product, wherein：First Analyte includes digoxigenin labeled and rhodamine marks；Second analyte includes rhodamine label and fluorescein label；First catches Obtain the antibody that reagent is anti-digoxigenin labeled；Second capture agent is the antibody of anti-rhodamine label；Third capture agent is made a living Object element resists fluorescein-labeled antibody；And the colloidal gold that signal interaction unit is Streptavidin coating.
Description of the drawings
The representative detection that Fig. 1 especially explanations carry out two kinds of analytes with single signal.
The representative detection that Fig. 2 especially explanations carry out three kinds of analytes with single signal.
Fig. 3 especially illustrates the two kinds of amplified productions just detected with colloidal gold.
Fig. 4 especially illustrates multicomponent bridge-jointing unit.
Fig. 5 especially illustrates the representative detection carried out using multicomponent bridge-jointing unit with single signal to two kinds of analytes.
Fig. 6 especially illustrates to be incorporated into the component of existing bridge-jointing unit only when multiple analytes are present in compound Detecting signal unit.
Fig. 7 especially explanations detect the nonrestrictive workflow of multiple analytes with single signal.
Fig. 8 describes the perspective view of representative device according to certain embodiments of the present invention.
Fig. 9 describes some components of representative device according to certain embodiments of the present invention.
Figure 10 describes some components of representative device according to certain embodiments of the present invention.
Figure 11 describes some components of representative device according to certain embodiments of the present invention.
Figure 12 describes some components of representative device according to certain embodiments of the present invention in various positions.
Figure 13：Describe the side view of some components of representative device according to certain embodiments of the present invention.
Figure 14 describes the side view of some components of representative device according to certain embodiments of the present invention.
Figure 15 A describe the side view of some components of representative device according to certain embodiments of the present invention.
Some components that Figure 15 B describe representative device according to certain embodiments of the present invention (such as but are not limited to non- Flexible connecting member) view.
Figure 15 C describe the perspective view of representative device according to certain embodiments of the present invention.
Figure 15 D describe the perspective view of representative device according to certain embodiments of the present invention.
Figure 16 describes the flexible connecting member for being connected to bonding pad.
Figure 17 describes the film in representative casing component.
Figure 18 describes the side view and top view of representative device according to certain embodiments of the present invention.
Figure 19 describes a kind of analysis analyte detection membrane system for representative device according to certain embodiments of the present invention System.
Figure 20 describes a kind of analysis analyte detection membrane system for representative device according to certain embodiments of the present invention System.
Figure 21 describes a kind of analysis analyte detection membrane system for representative device according to certain embodiments of the present invention System.
Figure 22 describes a kind of analysis analyte detection membrane system for representative device according to certain embodiments of the present invention System.
Figure 23 describes for the representative force member of representative device according to certain embodiments of the present invention.
Figure 24 A- Figure 24 D describe representative device according to certain embodiments of the present invention.
Figure 25 A- Figure 25 C describe representative device according to certain embodiments of the present invention.
Figure 26 describes representative device according to certain embodiments of the present invention.
Figure 27 A- Figure 27 B describe the view of representative device according to certain embodiments of the present invention.
Figure 28 describes the bottom view of representative device according to certain embodiments of the present invention.
Figure 29 describes the exploded view of representative device according to certain embodiments of the present invention.
Figure 30 describes the internal view of representative device according to certain embodiments of the present invention.
Figure 31 A- Figure 31 B describe the sectional view of representative device according to certain embodiments of the present invention.
Figure 32 describes the exploded view of representative device according to certain embodiments of the present invention.
Figure 33 describes the internal view of representative device according to certain embodiments of the present invention.
Figure 34 describes the sectional view of representative device according to certain embodiments of the present invention.
The representativeness that Figure 35 describes according to certain embodiments of the present invention moves locking component.
Figure 36 describes representative shell according to certain embodiments of the present invention.
Figure 37 describes representative shell according to certain embodiments of the present invention.
Figure 38 A describe representative device according to certain embodiments of the present invention.
Figure 38 B describe representative device according to certain embodiments of the present invention.
Figure 39 describes the enlarged drawing of representative device according to certain embodiments of the present invention.
Figure 40 describes the exploded view of cylinder and analysis analyte detection membranous system according to certain embodiments of the present invention.
Figure 41 describes representative device according to certain embodiments of the present invention.
Figure 42 describes representative device according to certain embodiments of the present invention.
Figure 43 A- Figure 43 C describe representative device according to certain embodiments of the present invention.
Figure 44 describes the exploded view of representative device according to certain embodiments of the present invention.
Figure 45 describes the exploded view of representative device according to certain embodiments of the present invention.
Specific embodiment
Describe provided herein is composition and method before, it should be understood that embodiment is not limited to described certain party Method, composition or method, because it can change.It should also be clear that term used in description is for some embodiment party of description The purpose of formula, and it is not intended to the scope of limitation embodiment.
Various methods and embodiment is described herein.Method and embodiment can be combined with each other.It is described herein fixed Justice and embodiment are not limited to ad hoc approach or example, it should so be limited unless the context clearly dictates.
As used herein, phrase " detection of analyte ", " detection and analysis object " refer to carry out a variety of analyses with single signal The detection of object.The detection of multiple analytes can be as described herein with single signal detection at least or just 2,3,4 or 5 Kind analyte.
It has to be noticed that as herein and used in following claims, it is unless the context clearly dictates otherwise, no Then singulative " one (a) ", " a kind of (an) " and " being somebody's turn to do (the) " refer to object including multiple.Unless otherwise defined, otherwise herein All technical and scientific terms used all have is generally understood identical meaning with those skilled in the art.Although class Any method for being similar to or being equivalent to approach described herein may be used to put into practice or test embodiments of the present invention, but Preferred method will now be described.All publication mentioned by this paper are all incorporated herein in entirety by reference, are incorporated to journey Degree supports described theme at present.It should in no way be construed so as recognizing that theme is had no right due to prior inventions prior in disclosure herein Hold.
As used herein, term " about " means ± the 10% of the numerical value of number be used therewith.Therefore, about 50% meaning Refer in the range of 45%-55%.In addition, phrase " about X to Y " is identical with " about X to about Y ", that is to say, that term " about " modifies " X " Both " Y ".
As used herein, term " optional " or " optionally " mean the structure, event or situation that then describe can with or can Not occur, and the situation that the situation for including event generation and event do not occur is described.
As used herein, term " sample " means that special article (such as analyte) or doubtful containing specific object can be contained Any fluid media (medium) or liquid of product.In some embodiments, the sample rich in dissolved solid can be used without into one Step processing, and in some embodiments, the sample containing high solid (non-dissolving) can be analyzed by using filter Or it is used with reference to other manual step.Sample in for approach described herein or device before or non-mistake It is filter or purifying.Sample can be liquid, suspension, extraction or the sample of dissolving or supercritical fluid.If sample will For flow device (perpendicularly or transversely (lateral)), then some flow behaviors be necessarily present in sample or extract with Allow to flow through apparatus described herein and system.The example of sample includes but is not limited to blood, food swab, food extraction Object, food suspension, food culture, bacterial cultures, viral cultures, amplified reaction, saliva, biofluid, PCR reactions Deng.Sample can also be obtained from another sample.For example, can to from another sample (such as food, cell, virus, Bacterium, blood etc.) mixtures of nucleic acids of extracting and developing and/or purifying carries out PCR reactions.PCR reactions will be considered as from another sample The sample that product obtain.
The food eaten raw or boiled that " food suspension " refers to place or be suspended in solution.Food solns can It is thinking mixing, vortex or blending." food culture " is the foodstuff samples cultivated under conditions of enriched sample.This Method is referred to as " being enriched with ".Enrichment can be used for promoting sample analysis preferably to detect multiple analytes with single signal Existence or non-existence.Sample may be the response sample derived from different samples.The example of response sample is " enrichment ".It lifts For example, blood or foodstuff samples through processing (for example, cultivate, purify, be separated into component etc.) and can be tested through locating The sample of reason is to detect multiple analytes.In some embodiments, two kinds of analyses are detected in blood sample or foodstuff samples Object.It in some embodiments, can be by carrying out two kinds of amplified reactions to two kinds of analyte tool specificity and then may be used To test and analyze object with single signal detection two kinds of amplified productions concurrently to detect the presence of two kinds of analytes in sample. In some embodiments, three kinds of analytes are detected using single signal.Detection can be parallel, that is to say, that only when all Signal is generated when analyte is present in same sample.It can be realized simultaneously by generating bridge joint compound described herein Row signal generates.The non-limiting embodiment of bridge joint compound is found in Fig. 1-Fig. 3.
As used herein, term " solid support " means essentially insoluble material in selected system or can Easily to detach the material of (for example, passing through precipitation) from the selected system that it is contained therein.Suitable for practice originally The solid support of inventive method can include activated or can activate to allow (such as the capture examination of certain compounds or molecule Agent, antibody etc.) it is incorporated into the group of solid support.Solid support can be, for example, agarose (agarose), agarose Gel (sepharose), polyacrylamide, agarose/polyacrylamide copolymer, glucan, cellulose, polypropylene, poly- carbon Acid esters, nitrocellulose, glassine paper or any other suitable substance for being capable of providing appropriate solid supporter.In some embodiment party In formula, solid support can be the form suitable for chromatographic particle, powder or gel.Solid support may be Film, nitrocellulose, PVC etc..Other kinds of film can also be used, and the type about the film that can be used has no Specific requirement.In some embodiments, solid support is test film.The example description of test film is in this article.
As used herein, term " analyte " includes but is not limited to antigen, by cell, virus, bacterium or other types Microorganism coding nucleic acid molecules, amplified production (such as amplicon), peptide, sugar etc..In some embodiments, analyte is not For antibody or its function fragment.It can be as described herein by using approach described herein and other known method Or the combinations such as device such as amplification method (such as PCR, RT-PCR etc.), hybridizing method, labeled primer detect nucleic acid molecules. Term " target molecule " can be used interchangeably with term " analyte ".Amplification method can be used for expanding nucleic acid present in sample point The amount of son is to promote the detection of analyte.The other kinds of analyte that approach described herein can be used to detect includes (but not limited to) antigen, antibody, receptor, ligand, chelate, protein, enzyme, nucleic acid, DNA, RNA, Insecticides (tech) ＆ Herbicides (tech), nothing Machine or organic compound or any material for caning be found that specificity combinating reagent.Analyte, which can also refer to, is present in same egg Different epitopes in white matter or polypeptide.Analyte can also refer to the analyte from cause of disease or nonpathogenic organism body.Analyte The analyte of interest being properly termed as in sample.That is, analyte is properly termed as just determining to exist by user or not depositing The reagent being in sample.
As discussed herein, analyte can be amplified production, such as the product of PCR reactions.PCR product is amplification to test oneself The nucleic acid sequence of test agent.Therefore, it detects the PCR product in sample and is to determine that the nucleic acid sequence on the basis as PCR product is It is no to be present in initial sample.For example, if whether those skilled in the art is just determining foodstuff samples by large intestine Bacillus pollutes, then can expand (for example, passing through PCR) to the nucleic acid sequence of Escherichia coli tool specificity and then according to herein Described method is detected.The detection instruction of amplified production (that is, amplicon), foodstuff samples contain has spy to Escherichia coli The native sequence nucleic acid of the opposite sex.This example is non-limiting and can be used for detecting other nucleic acid present in Natural Samples Sequence or other kinds of analyte.Analyte can be present in initial sample or for by using PCR from initial The analyte that sample obtains.When it is positive according to provided herein is method detect multiple analytes with single signal when, analyte also may be used With with heterologous label (tag) or interaction unit, and modified analyte is also referred to as analyte.In some embodiments In, analyte will be free of heterologous interaction unit, such as fluorescence labels, biotin, digoxin.
Analyte is different from the present or absent reagent for testing and analyzing object.Therefore, it adds in sample to determine The reagent that analyte whether there is is not analyte of interest.For example, in typical sandwich assay, the first antibody is connected to Solid support.Be coated with the solid support of antibody and sample contact with determine to be incorporated into antibody antigen presence or do not deposit .Then secondary antibody (secondary antibody) is additionally added to detect antigen.Then usually by adding in three-level antibody (third antibody, third antibody) detects the presence of secondary antibody, and three-level antibody, which has, is for example incorporated into its enzyme, make It (such as antibody of HRP connections) can be detected by various modes.Secondary antibody is not analyte of interest, because It is the reagent for detecting primary antigen (primary antigen).Therefore, sandwich assay will not be according to described herein Method detects the presence of multiple analytes with single signal, because secondary antibody is to detect depositing for antigen or analyte of interest Or the reagent or tool that are not present.Analyte is nor be found in bridge joint component physically or part.For example, in U.S.'s public affairs Open in application case US 2010/0273145, Fig. 1 and Fig. 2 show that analyte is incorporated into bridge joint entity, bridge joint entity later in conjunction in (signaling) entity signal to test and analyze the presence of object.Bridge-jointing unit or its any part or signalling entity are not It is analyte or analyte of interest.These components are the reagents for testing and analyzing object, in U.S. Published Application In the case of US2010/0273145, this is the detection of single analyte.U.S. Published Application US2010/0273145 about Thus the explanation of its figure and its component is herein incorporated by reference.
In some embodiments, analyte is protein, such as pathogen protein.Pathogen protein refers to from disease The protein of substance.The example of pathogen includes but is not limited to virus, prokaryotes body and such as pathogenicity most eukaryotes, Such as unicellular Pathogenic organisms and many cells parasite.Pathogen can also include protozoal pathogens, including Life Cycle Its interim stage for intracellular pathogen.As used herein, term " intracellular pathogen " means virus or Pathogenic organisms, At least part of its reproduction or life cycle is present in host cell and generates wherein or cause generation pathogen protein Matter.Pathogen may be food-borne causal agent.
Bacterial pathogens including but not limited to such as bacterial disease originality gram-positive cocci, include but is not limited to： Pneumococcus, staphylococcus and streptococcus.Pathogenicity Gram-negative coccus includes but is not limited to：Meningococcus and leaching ball Bacterium.Pathogenicity intestines gram-Negative bacillus includes but is not limited to：Enterobacteria, pseudomonad, actinomyces, Aitken bacterium (eikenella), glander-like disease, salmonella, Shigella, haemophilus, chancroid, brucella, soil draw bacterium, yersinia genus Salmonella (pasteurella), Streptobacillus moniliformis (streptobacillus moniliformis), spirillum (spirilum), list Listeria monocytogenes (Listeria monocytogenes), erysipelothrix ruhsiopathiae (Erysipelothrix Rhusiopathiae), diphtheria, cholera, anthrax, fifth venereal disease (donovanosis) (granuloma inguinale (granuloma )) and Bartonella (bartonellosis) inguinale.Pathogenicity anaerobic bacteria including but not limited to cause lockjaw, Botulism (botulism), other clostridium diseases, pulmonary tuberculosis, leprosy and other mycobacterial diseases bacterium. Pathogenicity spirochetal diseases include but is not limited to：Syphilis, treponematosis, yaws, pinta and halstern's disease and Leptospirosis.Other infection include but is not limited to as caused by superior pathogenic bacterium and pathogenic epiphyte：Actinomyces Disease, nocardiosis, cryptococcosis, blastomycosis, histoplasmosis and coccidioidomycosis, candidiasis, aspergillosis, Mucor Disease, sporotrichosis, paracoccidioidomycosis, Podbielniak mycosis, torulopsosis, maduromycosis, chromomycosis (chromomycosis) and dermomycosis.Rickettsial infection includes but is not limited to rickettsia and rickettsia Disease.The example of mycoplasma and choamydiae infection includes but is not limited to：Eaton agent pneumonia, lymphogranuloma venereum (lymphogranuloma venereum), psittacosis and perinatal period choamydiae infection.Causative protozoa and worm (helminth) and thus infectiousness eucaryote includes but is not limited to：Amcbiasis, malaria, leishmaniasis, trypanosomiasis, Toxoplasmosis, pneumocystis pneumoniae (Pneumocystis carinii), babesiosis, giardiasis, trichinosis, silk Parasitosis, snail fever, nematode, fluke or leech (fluke) and tapeworm (band worm (tapeworm)) infection.Bacterium further include (but It is not limited to) Listeria, Escherichia coli, Campylobacter and Salmonella.In some embodiments, Escherichia coli are large intestine Bacillus 0157.
The example of virus includes but is not limited to HIV, A type, B-mode and hepatitis C, FIV, slow virus, pestivirus (pestiviruses), West Nile Virus, measles, smallpox, cowpox, Ebola virus, coronavirus etc..Other pathogens It is disclosed in Patent Application Publication US20080139494, which is incorporated herein by reference.
In some embodiments, pathogen is food-borne causal agent.Analyte can also exist on food-borne causal agent On.Food-borne causal agent is to cause the pathogen of disease (such as viral or bacillary after contaminated food is eaten ).Food does not cause disease directly in itself, but it is more specifically present in the food-borne causal agent on the food for causing disease Consumption.In some embodiments, food-borne causal agent is Escherichia coli, Listeria, Campylobacter or Salmonella. In some embodiments, analyte is selected from food-borne causal agent analyte.For example, food-borne causal agent analyte can be (but not limited to) is selected from Escherichia coli analyte, Listeria analyte, Campylobacter analyte or Salmonella analyte. In some embodiments, analyte is specific O- antigens.In some embodiments, O- antigens are bacillus coli antigens And/or Salmonella O- antigens and it can be used for Escherichia coli and Salmeterol fluticasone propionate.In some embodiments, it analyzes Object is flagellin antigen.In some embodiments, analyte is Campylobacter spp flagellin antigen.In some embodiments In, analyte is virulence factor gene, such as the shiga toxin gene expanded from pathogenic Escherichia coli or salmonella.One In a little embodiments, analyte is expanded via amplification method (such as PCR or RT-PCR) and then according to described herein The DNA or RNA sequence of method detection.
As described herein, analyte can be amplified production.Amplified production, such as PCR product (such as double stranded PCR products) Unit can have been marked.Can by using by two kinds interaction units label or primer in connection come into The generation of amplified production that row is marked with unit.In some embodiments, there are two types of different interactions are single by tool for analyte Member so that bridge joint compound can be assemblnig and may detect multiple analytes by detecting signal unit.
As used herein, term " detecting signal unit " means to determine that analyte whether there is in sample after testing In unit.Detecting signal unit can be any reagent or composition that can be after testing.In some embodiments, signal is examined It surveys unit and is connected to capture agent.Therefore, detecting signal unit, which can be used for detecting, is incorporated into catching for its specific binding partner Obtain the presence of reagent.Capture agent can directly can be further included comprising detection reagent or capture agent and be tried containing detection The particle of agent.In some embodiments, capture agent and/or particle include pigment (color), colloidal gold, radioactive labels, Fluorescence labels or chemiluminescent substrate (substrate).In some embodiments, detecting signal unit includes near-infrared or red Outer label or substrate.In some embodiments, detecting signal unit includes pigment, colloidal gold, radioactive labels, fluorescence labels Or chemiluminescent substrate.In some embodiments, detecting signal unit include nanocrystal, functionalized nano-particle, on Convert (up-converting) nano-particle, cadmium selenide/cadmium sulfide fusion nano-particle, quantum dot and can be in NIR spectra Luminous near-infrared (NIR) fluorogen or material (the such as (but not limited to) such as material of lanthanide series cluster and phthalocyanine, Yi Jiyou The light emitting diode of CuPc, PdPc and PtPc composition).In some embodiments, capture agent and/or particle are incorporated into signal Detection unit, such as (but not limited to) colloid gold, silver, radioactive labels, fluorescence labels or chemiluminescent substrate, near-infrared compound (such as substrate, molecule, particle) or infrared compound (such as substrate, molecule, particle), nano-particle, radioactive nano particle, Quantum dot, magnetic particle or enzyme.
Detecting signal unit may be such as virion, latex particle, lipid particle, fluorescent particles, near-infrared grain Son or infrared particle.As used herein, term " fluorescent particles " means the particle to shine in fluorescence spectrum.As used herein, Term " near-infrared particle " means the particle to shine near infrared spectrum.As used herein, term " infrared particle " means The particle to shine in infrared spectrum.In some embodiments, colloidal gold has about 20nm, about 30nm or about 40nm or about Diameter dimension in the range of 20-30nm, about 20-40nm, about 30-40nm or about 35-40nm.In some embodiments, grain Attached bag metal-containing alloy particle.In some embodiments, metal alloy particle has about 10 diameters for arriving about 200nm.Metal The example of alloy particle includes but is not limited to golden metal alloy particle, gold-silver bimetal particle, silver metal alloy particle, copper Alloy particle, cadmium-Se particle, palldium alloy particle, platinum alloy particle and lead nano-particle.
As discussed herein, signal detection can be incorporated into one kind in analyte.It is incorporated into the signal detection of analyte The non-limiting examples of unit are shown in Fig. 1.Fig. 1 is described in greater detail in this article, and display is combined by capture agent 50 In the detecting signal unit 60 of analyte 40.However, detecting signal unit can also be incorporated into the other parts of compound.Only when Any component that there will necessarily be when multiple analytes are present in compound all can be detecting signal unit binding partners. Usually (but not exclusively), this component is by for one kind in analyte, but may be the capture agent for being incorporated into analyte. In contrast, in some embodiments, detecting signal unit is more than being incorporated into the analyte combined with solid support, consolidate Body supporter or the capture agent for being directly combined to solid support (if present on solid support).For example, in Fig. 1 In, detecting signal unit will not be directly combined to solid support 10, capture agent 15 or analyte 20.Not by any specific reason By constraint, if detecting signal unit is directly combined with solid support 10, capture agent 15 or analyte 20, then method will There is provided false positive results, i.e., signal will multiple analytes must not in the presence of detect.For example, Fig. 6 illustrates to combine In the detecting signal unit of the component of multicomponent bridge-jointing unit.The embodiment of bridge-jointing unit and multicomponent bridge-jointing unit is herein In and for example with reference to Figure 4 and 5 describe.Fig. 6 illustrates detecting signal unit 60, and wherein its capture agent 50 is incorporated into bridge-jointing unit 30 Component.Bridge-jointing unit 30 includes particle 34, the first capture agent 31, the second capture agent 32 and third capture agent 33.Fig. 6 Illustrate to be incorporated into the detecting signal unit of the second capture agent 32.Only examination is captured when two kinds of analytes are present in compound Agent 32 will be present in compound.If capture agent 32 is not present, this means the bridge joint compound there is no multiple analytes. Therefore, when multiple analytes are present in compound detecting signal unit will only be compound a part, thus avoid vacation Positive findings.If two kinds of analytes are not present, then therefore capture agent 32 will be not a part for compound, and, will There is no the binding partners of detecting signal unit.Therefore, detecting signal unit is detectable only in the presence of multiple analytes. Therefore, in some embodiments, detecting signal unit be incorporated into only when multiple analytes there is also when existing any component. Other characteristics, the feature and structure feature of multicomponent bridge-jointing unit are also disclosed in herein and are apparent based on the present invention 's.
The example of the device of presently described method wherein can be used to be described in such as United States Patent (USP) US8,012,770, U.S. In state patent application case US13/360,528 (submissions on January 27th, 2012), PCT Publication case WO 2011/044574, each case with The mode of reference is incorporated by herein.However, presently described method can be used with a variety of devices or form, such as Porous plate, array, microarray or in " ELISA " type format.The example of device also describes in this article, but these examples are non- Restricted.Approach described herein can also be used in combination with cross-flow devices.In cross-flow devices, the different portions of device Office is in the same plane opposite with vertical current device.The non-limiting examples of cross-flow devices can be found in United States Patent (USP) US 6,485,982, US 6,818,455, US6,951,631, US 7,109,042, RE39, in 664 grades, each patent is to quote Mode be incorporated to.Cross-flow devices can pass through adjustment for approach described herein, because described method is to be directed to Vertical current device is described.In cross-flow devices, indicate that the region of positive or negative result can include and be incorporated into point Analyse a kind of capture agent in object.Bridge-jointing unit can reside in one of transverse flow region or before being added in device It is mixed with analyte, this can also be directed to other devices and solid support carries out.Detecting signal unit can also be incorporated to transverse direction It flows in one of region.Such as by the present invention it is clear that the type of device or solid support is not critical and method can be based on Example described herein and embodiment are adjusted.
As used herein, term " amplicon " means the amplification expanded by PCR reactions or other amplified reactions or method Product, such as nucleic acid molecules.As discussed herein, amplicon can be analyte.Amplicon can be double-stranded nucleic acid molecule.Expand Increase production object can either directly or indirectly by using antibody or other capture agent systems (including it is described herein those) To detect.Amplified production can also be detected by hybridizing method as described herein in whole or in part.Amplified production It can for example be prepared by RT-PCR or linear amplification.
In some embodiments, amplicon is PCR product.PCR reaction products (such as amplicon) labeled can make It is detectable that it, which is obtained, by another antibody or antibody sample system, and such as (but not limited to) biotin-avidin/strepto- is close BRDU labels, the inserting agent of marker DNA, labeled dNTPS with prime system system, system, haptens system, DNA etc. can also It is labeled situation for wherein PCR product.Analyte can such as (but not limited to) be nucleic acid (single-stranded or double-strand) And can with antibody or other capture agent systems as described herein those identify or detect.Nucleic acid molecules can be marked Note has biotin labeling or uses the detectable other kinds of label of approach described herein.Other examples of label include Fluorescent marker.Fluorescent marker can be such as fluorescein (such as luciferin isothiocyanates (FITC)), rhodamine (such as tetramethyl Base rhodamine (TAMRA)) etc..Amplicon can be generated by using labeled primer with these labels.Label can lead to Cross the part that amplification program is incorporated in amplicon and therefore becomes analyte.Label will be considered as heterologous label, because should Label is not found in the native sequences of the template as amplicon.This paper institutes can be helped to be formed using label is incorporated into The capture agent (such as antibody) of the compound of description makes it possible to detect multiple analytes with single signal.These labels can For use as interaction unit.How Fig. 3 displays label can be used as interaction unit so that can be examined with single signal Survey the non-limiting examples of multiple analytes.
For example, in one embodiment, with the haptens and/or biotin labeling with analyte nucleic acid sequence homology DNA or RNA primers carry out PCR reactions.Analyte nucleic acid sequence can be toxin gene of the (but not limited to) from meat sample And/or lps molecule (such as shiga toxin).However, sample can be any sample, and analyte can be described herein Any other type analyte.PCR reactions can be carried out to generate the multiple analytes with interaction unit.With After primer amplification, approach described herein can be used to detect PCR samples.Can also use digoxin and/or TAMRA and/or PCR reactions are carried out with the FITC and TAMRA primers marked.These amplicons of label that can create a difference, amplicon can lead to It crosses and enables to detect multiple analytes with single signal together using capture agent bridge joint.The example of this kind of compound is shown It is shown in Figure 3.
Fig. 3 illustrate with anti digoxin antibody (that is, capture agent 15), digoxin/TAMRA label amplicon (that is, the One analyte 20, first interaction unit 21 and second interacts unit 22), anti-rhodamine antibody is ((that is, bridge-jointing unit 30), the amplicon of FITC/TAMRA labels is (that is, the interaction interaction of unit 41 and second of the second analyte 40, first is single The test film (that is, solid support 10) of member 42)；It is answered with the Streptavidin-gold for being incorporated into biotinylated anti-FITC antibody Close object (that is, signal generation unit 60 and third capture agent 50).
Briefly, after PCR reactions are carried out, amplicon can be with solid support, bridge-jointing unit and signal detection list Member contact.Solid support can have the capture agent of interaction unit being incorporated into the first analyte.Bridge-jointing unit There can be or be the capture agent for the unit that interacts being incorporated into the first and second analytes so that be incorporated into the first He Interaction unit in second analyte makes analyte become compound altogether.Detecting signal unit can be incorporated into presence In the interaction unit in one of second analyte.Detecting signal unit can then emit detectable signal or signal inspection Surveying unit can be detected by adding another detecting system.For example, in figure 3, detecting signal unit is is incorporated into second point Analyse the capture agent (for example, antibody) of the interaction unit on object.Detecting signal unit is through biotinylated.It can be subsequent The presence of detecting signal unit is determined by adding Streptavidin.Streptavidin will be deposited only in conjunction in two kinds of analytes Compound.In figure 3 in shown non-limiting examples, Streptavidin is to use colloid gold label, and colloidal gold makes it possible to Enough detections.However, other labels or detecting system can be used for detecting Streptavidin.In vertical current device described herein Embodiment in, test film is the solid support for having capture agent, and bonding pad can include detecting signal unit Or the molecule of the combination of detection detecting signal unit and the interaction unit of the second analyte.
Fig. 7 illustrates nonrestrictive workflow procedure, and it is more that described program may be used in the detection of amplicon single signal Analyte is planted to detect the presence of analyte of interest in the sample.Foodstuff samples 7000 are analyzed to determine pathogenicity large intestine bar The existence or non-existence of bacterium.Foodstuff samples 7000 through processing (for example, enrichment, culture, nucleic acid, purifying, separation, extraction or other Similar step) with extracting and developing or otherwise obtain the nucleic acid being present in foodstuff samples.It is present in through handling sample Nucleic acid sequence in 7001 can expand, such as (but not limited to) by PCR, with specific amplification pathogenic Escherichia coli sequence Row.The example description of these sequences is in this article.It does not need to using specific primer group, because can be based on target sequence to be amplified Row modify those sequences.As described herein, primer can be labeled, and thus generating labeled amplicon (has heterologous phase The analyte of interaction unit).If target sequence is present in foodstuff samples and through handling in sample, then will generate the first analysis 7020 and second analyte 7040 of object.Show that analyte has heterologous interaction unit (7021,7022,7041 and 7042). Analyte can be mixed with bridge-jointing unit 7030.Mixture will form and bridge compound 7100.It can be then by answering bridge joint Object is closed to connect with the solid support 7010 comprising capture agent 7015 and the detecting signal unit 7060 comprising capture agent 7050 It touches to test and analyze object.As discussed herein, the detecting signal unit 7060 comprising capture agent 7050 can be absorbed in film Above and itself and bridge joint compound is allowed to interact.Solid support 7010 comprising capture agent 7015 can be with anti- The test film of body.These elements (element) can be incorporated in device as described herein.Although Fig. 7 step displays are point It does not carry out, but it can also be carried out with different order and some steps can combine.For example, make analyte and bridge joint The step of unit mixes can also with that analyte is made to contact with the detecting signal unit comprising capture agent is combined.It can be subsequent The detecting step being then added in compound to solid support.In some embodiments, analyte, bridge-jointing unit, Signal interaction unit comprising capture agent and the solid support comprising capture agent can be mixed simultaneously or almost simultaneously It is combined and subsequent detectable signal detection unit.Only letter is detected when multiple analytes are present in tested sample Number detection unit or (that is, more than negative control) detects more than background level.That is, in the figure 7, if will be only at two kinds Compound 7200 is formed when analyte and therefore two target sequences are present in foodstuff samples 7000.If in one of analyte Compound 7200 will not be formed during missing.The workflow shown in Fig. 7 can also include washing step and not formed again with washing away Close any unbonded material or component of object 7200.Washing step can also be incorporated herein in described any method.
In some embodiments of approach described herein, included with the method for single signal detection multiple analytes Amplification is present in multiple target nucleic acid sequences in sample.Target sequence can be analyte or amplification product can be analyte. Presence of the detection instruction template sequence of the sequence (for example, PCR product) of amplification in primary sample.
In some embodiments, the method that multiple analytes are concurrently detected with single signal includes a) using single letter The device of number detection multiple analytes is contacted with one or more samples for including multiple analytes；With detection detecting signal unit Existence or non-existence, the detecting signal unit concurrently indicate the first analyte of interest and of interest second analysis The existence or non-existence of object.Device can be present or absent any device for testing and analyzing object, including (but it is unlimited In) apparatus described herein.In some embodiments, device includes：Shell, it includes：It is contacted with bonding pad in fluid Import；Force member；Contact the slidably locking component of force member；Contact the connecting elements of force member；It connects The sliding button of component；With the detection membranous system comprising bonding pad, test film and absorption component, bonding pad, test film and absorption At least part of component is substantially parallel to each other, and force member contact detects membranous system and can apply substantially perpendicular to inspection The pressure of membranous system is surveyed, slidably locking component, bonding pad include the signal inspection comprising third capture agent for sliding button movement Survey unit；Test film includes the first capture agent for being fixed on test film.
In some embodiments, one or more samples include the first analyte of interest, of interest second point Analysis object and the bridge-jointing unit for including the second capture agent, wherein the first analyte of interest, which includes, is incorporated into the first capture agent The first interaction unit and be incorporated into the second interaction unit of bridge-jointing unit, and the second analyte packet of interest Containing the first interaction unit for combining bridge-jointing unit and the second interaction unit.In some embodiments, signal detection Unit includes third capture agent, and third capture agent is incorporated into the first interaction list of the second analyte, the second analyte Member or the second interaction unit, the first and second analytes compound component or only when compound contains first and the The component of existing bridge-jointing unit during two analytes.
In some embodiments, detection is included in the parts of one or more samples and has contacted and flowed through bonding pad Bonding pad is moved later, thus makes at least part exposure of test film for the detection of detecting signal unit, so as to use Single signal indicates the existence or non-existence of multiple analytes.In some embodiments, by making slidably locking component shifting It moves to move bonding pad.In some embodiments, one or more samples connect before compressed detected membranous system with bonding pad It touches.Method can be carried out detecting multiple analytes with multiple samples.For example, if it is multiple to generate to carry out multiple amplified reactions Amplicon (analyte), then multiple amplified reactions are respectively considered as independent sample.In order to detect a variety of analyses with single signal Object, sample must mix.Multiple samples can be mixed before device is contacted or sequentially or concurrently with device (solid support) Contact.
In some embodiments, the first and second analytes are amplicon.In some embodiments, first and second Analyte is PCR reaction products.In some embodiments, the first interaction unit of the first analyte is digoxin mark Note.In some embodiments, the second interaction unit of the first analyte is marked for rhodamine.In some embodiments In, the first interaction unit of the second analyte is marked for rhodamine.In some embodiments, the second of the second analyte The unit that interacts is marked for fluorescein.In some embodiments, third capture agent is incorporated into the second of the second analyte Interact unit.In some embodiments, third capture agent is biotinylated capture agent.In some embodiments In, signal interaction unit is coated with Streptavidin.In some embodiments, signal interaction unit is strepto- parent With the colloidal gold of element coating.In some embodiments, the first and second analytes are nucleic acid amplification product, wherein：First point It analyses object and includes digoxigenin labeled and rhodamine label；Second analyte includes rhodamine label and fluorescein label；First capture Reagent is anti-digoxigenin labeled antibody；Second capture agent is anti-rhodamine labelled antibody；Third capture agent is biotinylation Anti- fluorescein labelled antibody；And the colloidal gold that signal interaction unit is Streptavidin coating.
As herein and used in the whole text, term " (attached) of connection " or " connection (attachment) " can be included directly It connects or is indirectly connected in succession.Two components being connected to each other directly are also each other in physical contact.Two be indirectly connected with each other Component is connected by intermediate module.For example, if component A is directly connected in component C and component C is directly connected in component B, that Component A can be indirectly coupled to component B.Therefore, in this kind of example, component A will be referred to as being indirectly coupled to component B.
Term " capture agent " means that target molecule to be detected in sample or the reagent of analyte can be combined.Capture agent Example include but is not limited to antibody or its antigen-binding fragment, oligonucleotides and class peptide.Other example packets of capture agent Include (but not limited to) small molecule or protein, as biotin, avidin, Streptavidin, haptens, digoxin, BRDU, single-stranded and double-strandednucleic acid binding protein or other inserting agents etc. identify and capture its molecule.These are capture agent Non-limiting examples.Other kinds of capture agent can also be used.
As discussed herein, capture agent can also refer to such as antibody.Complete antibody (also referred to as immunoglobulin) allusion quotation It is type four polyglycosylated albumen being made of two light (L) chain of each about 25kDa and two weight (H) chains of Ge Yue 50kDa.Two The light chain (being known as λ and κ) of type is present in antibody.The amino acid sequence of constant domain depending on heavy chain, will be immune Globulin is assigned as five primary categories：A, D, E, G and M, and some in these classifications can be further divided into subclass (together Kind type), such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2.Each light chain is by variable (V) structural domain (VL) of N-terminal and constant (C) structural domain (CL) forms.Each heavy chain is by a N-terminal V structure domain (VH), three or four C-structure domains (CH) and a hinge District's groups into.CH structural domains closest to VH are designated as CH1.The relatively conservative sequence that VH and VL structural domains are known as framework region by four Column region (FR1, FR2, FR3 and FR4) forms, these regions form three high variable sequence region (complementary determining region, CDR) Skeleton.CDR contains most of residue of the specificity interaction of responsible antibody or antigen-binding proteins and antigen.CDR is claimed For CDR1, CDR2 and CDR3.Therefore, the CDR components on heavy chain are referred to as H1, H2 and H3, and the CDR components on light chain are referred to as L1, L2 and L3.CDR3 is the largest source of molecular diversity in antibody or antigen-binding proteins binding site.For example, H3 It may be as little to two amino acid residues or more than 26 amino acid.The subunit structure and three-dimensional of different classes of immunoglobulin Construction is well-known in the art.About the summary of antibody structure, referring to antibody：Laboratory manual (Antibodies:A Laboratory Manual), cold spring harbor laboratory (Cold Spring Harbor Laboratory), Ha Luo (Harlow) et al. volume, 1988.Those skilled in the art should be understood that each subunit structure, for example, CH, VH, CL, VL, CDR and/or FR structure, comprising active fragment.For example, active fragment can be sub- single by combining VH, VL or CDR of antigen Position part (that is, antigen-binding fragment) or be incorporated into and/or activate Fc receptors and/or complement CH subunits part group Into.
The non-limiting examples of binding fragment covered in the term as used herein " antigen-specific antibodies " include：(i) Fab segments, the monovalent fragment being made of VL, VH, CL and CH1 structural domain；(ii) 2 segments of F (ab'), comprising passing through hinge area The bivalent fragment of the two Fab segments of disulfide bridge bond even；(iii) the Fd segments being made of VH and CH1 structural domains；(iv) by antibody The Fv segments of the VL and VH structural domains composition of single armed；(v) dAb segments are made of VH structural domains；The CDR of (vi) separation.This Outside, although two structural domains VL and VH of Fv segments are encoded by separate gene, can by synthetic linker (linker, Connexon) recombination engagement, it generates wherein VL and VH structural domains pairing and (is known as scFv to form the single protein chain of monovalent molecule (scFv)).Most common connector is 15 residue (Gly4Ser)3Peptide, but it is also known that other connectors in fields.It is single-stranded anti- Body is also intended in " antigen-binding fragment " for being covered by term " antibody or antigen-binding proteins " or antibody.Antibody may be more Clonal antibody, monoclonal antibody, chimeric antibody, antigen-binding fragment, Fc segments, single-chain antibody or its any derivative.Capture Reagent or antibody may be VHH areas, bispecific antibody, the peptide fragment comprising antigen binding site or is incorporated into of interest The compound of antigen.Antigen of interest can be amplicon or other kinds of analyte.
These antibody are commercially available or obtained using routine techniques known to those skilled in the art, and with it is complete The identical mode of antibody screens the effectiveness of these segments.Germ line genes and multiple body cells by a variety of coding variable domains Event generates antibody diversity.Somatic events include variable gene segment and diversity (D) and engage (J) constant gene segment C Recombination prepares complete VL structural domains to prepare complete VH structural domains and variable and engagement constant gene segment C recombination.Weight Group process sheet causes the amino acid at V (D) J tie points to lose or addition as inaccurate.Before antigen exposure, these Diversity mechanism is come across in developing B cell.After antigenic stimulus, the antibody gene experience body expressed in B cell is thin Cytoplasmic process becomes.The random recombination of estimated number, these sections based on germline gene segment and random VH-VL pairings, can generate Up to 1.6x107Kind different antibodies (basic immunology (Fundamental Immunology), the 3rd edition (1993), Borrow (Paul) it compiles, crow publishing house (Raven Press), New York (New York), N.Y.).When consideration contributes to antibody diversity During other processes of (for example, somatic mutation), it is believed that can generate more than 1x1010Kind different antibodies (immunoglobulin gene (Immunoglobulin Genes), second edition (1995), Ji Aoniao (Jonio) et al. volume, academic press (Academic Press), Santiago (San Diego), California (Calif.)).Since many processes participation generation antibody is various Property, the independent derivative monoclonal antibody with same antigen specificity would be impossible to same amino acid sequence.
Can by the well-known method of those skilled in the art come generate can with antigen described herein, Epitope or the antibody or antigen-binding proteins molecule of the interaction of other molecular specificities.It for example, can be by according to Perception method generates hybridoma to generate monoclonal antibody.Standard method, such as Enzyme-linked Immunosorbent Assay point can then be used Analysis (ELISA) and Biacore, which are analyzed, screens hybridoma formed in this way, with identify one or more generations and The hybridoma of molecule of interest or the antibody of compound specificity interaction.
As the alternative solution for the hybridoma for preparing secrete monoclonal antibody, can be screened by using polypeptide of the present invention Recombination combination immunoglobin libraries (for example, antibody phage display library) are incorporated into the immune globulin of polypeptide thus to detach Text of an annotated book library member identifies and detaches the monoclonal antibody of polypeptide of the present invention.For generating and screening the skill of phage display library Art and commercial reagent box are well-known for those skilled in the art.In addition, it is particularly useful for generation and screening antibodies Or the method for antigen-binding proteins display libraries and the example of reagent can be found in document.
Term " capture agent " also includes chimeric antibody, such as humanized antibody and full-length human antibody.At some In embodiment, capture agent is the anti-colon bacillus 0157 of goat:H7 antibody catalog number (Cat.No.)：70-XG13 (luxuriant and rich with fragrance hereby Gerald industry (Fitzgerald Industries))；Colon bacillus 0157:H7 monoclonal antibody catalog number (Cat.No.)s：10-E13A (luxuriant and rich with fragrance hereby Geralds Industry)；Colon bacillus 0157:H7 catalog number (Cat.No.)s：10C-CR1295M3 (luxuriant and rich with fragrance hereby Gerald industry)；Colon bacillus 0157:H7 is mono- Clonal antibody catalog number (Cat.No.)：10-E12A (luxuriant and rich with fragrance hereby Gerald industry)；Or goat anti-mouse IgG catalog number (Cat.No.)：ABSE-020(DCN). Capture agent may be such as albumin A, Protein G.Capture agent may be to combine or specifically bind to fluorescent marker The antibody of (for example, fluorescein or rhodamine), haptens, digoxin etc..Capture agent such as Streptavidin can be with colloid Gold combines.Streptavidin-Au composite can be for subsequent use in being for example incorporated into biotinylation product, such as biotinylation resists Body.Non-limiting examples are found in Fig. 3.Being merely to illustrate property of label purpose shown in Fig. 3 and other rows can be used Row.
Capture agent can also include antiantibody, that is, identify another antibody but do not have the antibody of specificity, example to analyte Such as (but not limited to) anti-igg, anti-IgM or anti-IgE antibodies.
As used herein, term " concurrently " refers to simultaneously or almost simultaneously detect multiple analytes.As used herein, " method that multiple analytes are concurrently detected with single signal " or its version refer to using it is single analysis (for example, single hole, Single-point, the single position on an array) or special purpose device come with single signal detection multiple analytes method. If different devices, hole or array are used for by same signal detection multiple analytes, then this is not parallel with single signal The method that ground detects multiple analytes.For a kind of method to as the side that multiple analytes are concurrently detected with single signal Method, the method must generate single only at single position (hole, point, line on film or other kinds of solid support etc.) Signal (the example description of signal is in this article), this informs that user's multiple analytes are present in sample.For example, by same letter Number for indicate in different holes single analyte whether there is in this hole (or point on array) and with post analysis it is multiple To determine that multiple analytes whether there is, this is not the method that multiple analytes are concurrently detected with single signal in hole (or point).
As used herein, term " single signal " means the signal detection based on single part or method.It is if for example, single One signal is color red, then is based only upon the presence of color red to indicate multiple analytes.That is, in this nonrestrictive reality In example, color red instruction multiple analytes are present in sample.In contrast, if a kind of analyte is referred to by color red Show and the second analyte indicated by color yellow, then using two kinds of colors (that is, signal) be not with single signal detect it is more Kind analyte.Signal is not limited to colorimetric detection.The example of workable signal provided herein.
Term " detection (detecting) " or " detection (detection) " are analyzed with most broad sense using to include target The qualitative and/or quantitative measurment of object.
As used herein, term " interaction unit " means analyte or is connected to the heterologous label or tag of analyte A part, by another molecule (for example, capture agent, bridge-jointing unit or detecting signal unit) identify or combine.Phase interaction It can be analyte a part in itself with unit or can be heterologous label or tag.Interaction unit may be identification The antibody of analyte or other kinds of capture agent.In some embodiments, analyte can comprise more than 1,2,3,4 or 5 interaction units.In some embodiments, interaction unit be incorporated into be present in analyte in itself or as point Analyse the capture agent of another interaction unit on the heterologous label of a part for object.If for example, analyte be peptide or A part for protein a, then part or peptide for protein can be by capture agent, bridge-jointing unit or signal detection in itself The interaction unit of unit identification.In some embodiments, peptide can also be covalently attached to heterologous label or tag and different The compound of source label or tag or peptide and heterologous label or tag is considered as the unit that interacts.Therefore, in some embodiment party In formula, analyte includes the first interaction unit and/or the second interaction unit.In some embodiments, one or Multiple interaction units can be analyte inherently or one or more interaction unit can by some its His method, such as crosslinking, by chemically react be covalently attached, noncovalent interaction is (for example, antibody-antigene, one in analyte Part and hybridizing between another molecule) it adds.It is (for example, two nucleotide by two kinds of molecules in interaction unit Sequence) hybridization when being formed so that the unit that interacts will be considered as by the part of another molecular recognition in hybrid product.Mutually Action cell can also be added to by amplified reaction in analyte.This can be by using the primer containing interaction unit To generate.The unit that interacts can also have detectable signal, but it is not these signals to be detected.
As used herein, the term " heterologous " about interaction unit mean the natural all groups of not analyte, Molecule or part.For example, amplified production can only include nucleic acid molecules or nucleotide base.However, amplified production can combine In or be connected to heterologous label, such as (but not limited to) haptens, biotin, digoxin, fluorescent molecular (if for example, fluorescein or It is red bright) etc..The example of heterologous interaction unit includes but is not limited to haptens, biotin, nucleic acid molecules, peptide fragment (example Such as His labels, GST labels), enzyme, Streptavidin, avidin, fluorescent molecular etc..This inventory is non-limiting And can use any interaction unit.Analyte can use such as digoxin, rhodamine, fluorescein, DNP, BRDU Molecular labeling and then by having the capture agent of specificity to detect to given molecule.
According to some embodiments, the method that detection multiple analytes are provided.Presently described method can be used for detecting Multiple analytes.It is unexpected and surprising result is that, can use single signal detect multiple analytes.This has Unpredictable consequence, you can only to detect the existence or non-existence of multiple analytes by a kind of detection of signal.This with The presence of multiple analytes in sample is detected using unlike signal or need to be detected the only of multiple analytes in same reaction Vertical reaction and method are contrasted to detect the presence of multiple analytes.That is, embodiments described herein partly provides The method that multiple analytes are concurrently detected with single signal so that multiple analytes in the detection instruction sample of single signal In the presence of or single signal there is no multiple analytes in instruction sample to be not present.Embodiment of the present invention is provided with single The method for detecting to signal parallel at least 2,3,4 or 5 kind of analyte.In some embodiments, method can be used for single Detect to signal parallel 2,3,4 or 5 kind of different analyte.Although providing multiple examples about 2 kinds of analytes of detection, can with Based on the present invention is about 3,4 or 5 kind of analyte is adjusted and amending method.
As used herein, term " different analytes " means that analyte differs.However, different analytes can be the same Title refers to, but the different strains from different organisms or from same organism.For example, different organisms contain with identical The gene and protein of function, and therefore it is given same name.But gene or protein are to come from separate sources and therefore quilt It is considered as different analytes.They may or may not have different sequences.Different analytes can also mean to come from different biologies The analyte of body.For example, a variety of bacterial strains there are Escherichia coli.Not all bacterial strains of Escherichia coli cause food-borne disease Disease.The method of the present invention can be used for for example detecting the multiple analytes from pathogenic escherichia coli strains, such as be come from detection The analyte of avirulence coli strain compares.Although it can be mentioned that certain types of analyte, divides in the present invention Analyse object can be any kind of analyte, analyte classification such as (but not limited to) described herein.
For example, in some embodiments, provide the method for concurrently detecting the first analyte and the second analyte.One In a little embodiments, method includes making the solid support comprising the first capture agent and the first analyte, the second analyte, packet Bridge-jointing unit containing the second capture agent and the detecting signal unit contact comprising third capture agent；With detection signal detection list The presence of member, detecting signal unit indicate the presence of the first analyte and the second analyte.In some embodiments, it first catches It obtains reagent and is fixed on solid support.In some embodiments, the first analyte, which includes, is incorporated into the of the first capture agent One interaction unit and the second interaction unit for being incorporated into bridge-jointing unit；Second analyte is included with reference to bridge-jointing unit First interaction unit and the second interaction unit for being incorporated into detecting signal unit.Signal detection list can then be detected Member.If detect detecting signal unit, then it indicates that multiple analytes are existing.
It is not intended to be bound by any theory, can concurrently detect multiple analytes by forming compound.In some realities It applies in mode, compound includes solid support, the first analyte, the second analyte, bridge-jointing unit and detecting signal unit, Each member of middle compound is directly or indirectly bonded to each other.Sample can be washed, while retains solid support, and compound Object will be detected only when forming compound.The example of these compounds is found in Fig. 1-Fig. 3, is further added herein With description.
In some embodiments, the method for concurrently detecting the first analyte and the second analyte is provided, method includes： Make solid support and the first analyte of interest, the second analyte of interest, the bridge joint list comprising the second capture agent Member and the detecting signal unit contact comprising third capture agent；With the existence or non-existence of detection detecting signal unit, signal Detection unit concurrently indicates the existence or non-existence of the first analyte of interest and the second analyte of interest, wherein： First capture agent is fixed on solid support；First analyte of interest includes the first phase for being incorporated into the first capture agent Interaction unit and the second interaction unit for being incorporated into bridge-jointing unit；And the second analyte of interest is included to combine and be bridged First interaction unit of unit；It is incorporated into the second analyte, the first interaction unit of the second analyte or the second phase Interaction unit, the compound of the first and second analytes or the existing bridge only when compound contains the first and second analytes The detecting signal unit of the component of order member.
Fig. 1 explanations can form compound concurrently to detect two kinds of analytes with single signal.Fig. 1 explanations are fixed on The capture agent 15 of body supporter 10.Capture agent 15 is incorporated into the first analyte 20.Bridge-jointing unit 30 is incorporated into the first analysis Object.Bridge-jointing unit is herein in connection in the second analyte 40.Fig. 1 also illustrates comprising the capture agent 50 for being incorporated into the second analyte 40 Detecting signal unit 60.Fig. 1 illustrates that this compound is only formed when each member exists and can be bonded to each other.It then can be with Detect detecting signal unit.Fig. 3 also shows the embodiment that two kinds of analytes are detected with single signal.Fig. 3 display specificity marks Remember (such as FITC, TAMRA, DIG, biotin, Streptavidin etc..), but these labels can be according to the present invention through repairing Decorations.
In some embodiments, method includes one or more washing steps.Washing step, which can be used for removing, not to be tied The material of conjunction.For example, if solid support is contacted with the first sample, then solid support can be washed any to remove Unbonded material.Solid support for bead (bead) some embodiments in, bead can be contacted with sample and with After can wash the bead.Washing bead is conventional and those skilled in the art is well-known.It can be based on institute Usually it is not that bead or other kinds of solid support are washed in the particular solid supporter change of key feature or selection Method.
In some embodiments, the sample with the first analyte is contacted with solid support.In some embodiments In, mixture is washed so that is not associated with no longer existing in any material of solid support.In some embodiments, Also the different samples with same sample or comprising the second analyte and/or bridge-jointing unit contact solid support.Mixture can be with Then wash to remove any unbonded material again.In some embodiments, signal of the addition comprising capture agent is examined Survey unit.It can also include washing step to remove any unbonded detecting signal unit.Signal detection can then be detected Unit or existing another reagent that detection detecting signal unit can be added.In some embodiments, all steps are same When or be almost carried out at the same time.During the progress of method, it can be inserted into washing step in due course.
In some embodiments, different analytes or sample can be mixed before or while solid support is applied to It is combined.Sample can be such as amplification reaction mixture, be used to generate or attempt to generate analyte.In some embodiment party In formula, different amplified reactions will be carried out to expand multiple analytes.Therefore, sample or analyte be applied to solid support it Before, sample or analyte can be mixed.Sample or analyte can also before being contacted with the surface of solids with capture Reagent and/or bridge-jointing unit mixing.
In some embodiments, the of the first and second interaction units of the first analyte and the second analyte One and second interaction unit be each independently heterologous interaction unit.In some embodiments, the first analyte Interaction unit and the second analyte interaction unit be haptens.In some embodiments, the first analyte Interaction unit with the second analyte is fluorescein or rhodamine molecule.Therefore, in some embodiments, the first analysis Object and the second analyte have at least one common interaction unit.Interacting the concomitant of unit will be so that bridge joint be single Member can make two kinds of analytes become detectable compound.In some embodiments, the first and second analytes do not have Identical interaction unit.In this case, for some embodiments, bridge joint entity will be that can make two kinds of analytes The divalent capture agent (for example, bivalent antibody) of key even each other.Divalent capture agent will be in combination in first and second Analyte.In some embodiments, each interaction unit being present in multiple analytes is different.In some implementations In mode, some interaction units are different, but some interaction units are identical.In some embodiments, Analyte includes haptens interaction unit and biotin interaction unit.In some embodiments, the first analyte Include digoxin interaction unit and rhodamine interaction unit；Second analyte include rhodamine interaction unit and FITC interaction units, and bridge-jointing unit is incorporated into rhodamine interaction unit.Bridge-jointing unit, which can subsequently form, to be contained There is the compound of the first and second analytes.This is found in such as Fig. 3.
In some embodiments, multiple analytes are same type of analytes.For example, each analyte detected can Think peptide.In some embodiments, each analyte is nucleic acid molecules, such as amplified production (for example, amplicon).Analyte Can be any types, analyte including but not limited to described herein.In some embodiments, analyte is different 's.In some embodiments, the first analyte is amplified production and the second analyte is protein or peptide.Analysis can be used Any combinations of object.
As used herein, term " bridge-jointing unit " or " bridge " mean to make two or more analyte in compound The one or more molecules of middle key even.I.e., for example, bridge-jointing unit can be incorporated into the interaction list in the first analyte Interaction unit in member and the second analyte.If only detecting two kinds of analytes, a kind of bridge-jointing unit can be used.Such as Fruit detects three kinds of analytes, then can use two kinds of bridge-jointing units.In some embodiments, method uses " n-1 " kind bridge joint Unit, wherein " n " is the number of detected analyte.In some embodiments, it is detected using single bridge-jointing unit super Cross 2 kinds of analytes.The example of bridge-jointing unit includes but is not limited to immunoglobulin molecules (such as IgM, IgE, IgG, IgA Deng), Streptavidin and bridge-jointing unit is allowd to be incorporated into the unit of the interaction more than one comprising a variety of capture agents Molecule.In some embodiments, bridge-jointing unit is multivalence capture agent.
In some embodiments, bridge-jointing unit is the compound of compound, substance or macromolecular.For example, bridge-jointing unit It can include and be coated with the nano-particle of antibody and another antibody.In this non-limiting examples, it is coated with the nanometer of antibody Particle, which can contain the antibody for being incorporated into analyte or the interaction unit in analyte and also contain, is incorporated into another resist The antibody of body.Another antibody can be incorporated into different analytes.It is coated with the nano-particle of antibody and the phase interaction of another antibody It is bridged together with will then make different analytes.The non-limitative illustration of this bridge joint compound can see Fig. 4 In Fig. 5, hereinafter also it is described.Other versions of the bridge as compound can also be prepared.The essence of bridge-jointing unit True structure and form are not necessary, as long as it can be by multiple analytes " bridge joint " in the composite.Therefore, Ke Yiyou Multiple subunits or component form bridge so that analyte to be bridged together.Although bridge-jointing unit can be bridged through illustrating and being discussed as Two kinds of analytes, but bridge-jointing unit can be designed to bridge more than 2 kinds analytes, such as 3,4,5 kind or more kind.Therefore, In some embodiments, bridge-jointing unit bridge joint 2,3,4,5 kind or more kind analyte.In some embodiments, bridge joint is single Member bridge joint at least 2,3,4 or 5 kind of analyte.The non-limiting examples of 2 kinds of analytes are bridged merely to reaching illustrative purpose And embodiments disclosed herein is not limited to only bridge 2 kinds of analytes.
As discussed herein, more than 2 kinds analytes can be detected using the method for the present invention.For example, offer single signal The method for concurrently detecting the first analyte, the second analyte and third analyte.Detection for other analyte, can be with Similar fashion adapting method.In some embodiments, method includes making first, second, and third analyte and solid support Object, the first bridge-jointing unit, the second bridge-jointing unit and detecting signal unit contact；With the presence of detection detecting signal unit, signal Detection unit concurrently indicates the presence of first, second, and third analyte with single signal, wherein the first analyte includes the One interaction unit and the second interaction unit；Second analyte includes the first interaction unit and the second interaction Unit；Third analyte includes the first interaction unit and the second interaction unit；Solid support, which includes, is incorporated into the First capture agent of the first interaction unit of one analyte；First bridge-jointing unit is incorporated into the second phase of the first analyte First interaction unit of interaction unit and the second analyte；Second bridge-jointing unit is incorporated into the of the second analysis substance markers The third interaction unit of two interaction units and third analyte；And detecting signal unit is incorporated into third analyte Second interaction unit.It is not bound by any theory, it is contemplated that object can be concurrently tested and analyzed, because of first, second He Third analyte forms compound, and wherein compound includes solid support, the first analyte, the second analyte, third analysis Each member of object, the first bridge-jointing unit, the second bridge-jointing unit and detecting signal unit, wherein compound ties directly or indirectly to one another It closes.
Fig. 2 illustrates the compound of three kinds of analytes of the detection that can be formed, and is similar to example illustrated in fig. 1.Fig. 2 is said The bright solid support 10 with the capture agent 15 for being incorporated into the first analyte 70.First analyte is incorporated into the first bridge joint list Member 80.First bridge-jointing unit is incorporated into the second analyte 20, is also incorporated into the second bridge-jointing unit 30.Second bridge-jointing unit is also tied Together in third analyte 40, it is incorporated into capture agent 50.Capture agent is also connected to detecting signal unit 60.Therefore, Fig. 2 is said The bright non-limiting examples that three kinds of analytes how can be detected with single signal.
Fig. 4 illustrates the nonrestrictive bridge joint compound by being more than a kind of molecule, macromolecular or material composition.This compound It is properly termed as multicomponent bridge joint compound.Fig. 4 explanations include particle 34, the first capture agent 31, the second capture agent 32 and the The bridge-jointing unit 30 of three capture agents 33.Bridge-jointing unit 30 can make the first analyte 20 and the second analyte 40 altogether and shape Bonding connects the compound of the first analyte 20 and the second analyte 40.Fig. 4 illustrates particle 34 (for example, nano-particle, polyphenyl second Alkene, agarose etc.), particle is coated with the first capture for being incorporated into the first analyte 20 indirectly directly or by interaction unit Reagent 31 exists in third capture agent 33 on particle 34, and third capture agent is incorporated into be combined with the second analyte 40 The second capture agent 32.Can be then according to approach described herein and combination this compound of analyte detection, this is illustrated in In Fig. 5.Fig. 5 shows the bridge joint compound of Fig. 4, compound and the solid with the capture agent 15 for being incorporated into the first analyte 20 Supporter 10 and detecting signal unit 60 comprising the capture agent 50 for being incorporated into the second analyte 40 interact.Such as this paper institutes It discusses, the explanation for being incorporated into the detecting signal unit of the second analyte is merely for illustrative purpose.Detecting signal unit also may be used To combine the other parts of compound, if detecting signal unit be not incorporated into the analyte that interacts with solid support or Solid support is in itself.Solid support 40 can be the test film shown in such as test film, such as Fig. 3.Herein Other examples of solid support are provided.
The present invention is provided comprising solid support, the first analyte, the second analyte, bridge-jointing unit and detecting signal unit Compound, each member of wherein compound combines directly or indirectly to one another.In some embodiments, solid support knot Together in the first analyte, bridge-jointing unit is incorporated into the first analyte and the second analyte, and detecting signal unit is incorporated into Two analytes.In some embodiments, solid support includes the first capture agent, and the first analyte includes the first phase interaction With unit and the second interaction unit, the second analyte includes the first interaction unit and the second interaction unit, bridge Order member includes the of one or more the second interaction units for being independently incorporated into the first analyte and the second analyte The capture agent of one interaction unit, and detecting signal unit includes the second interaction list for being incorporated into the second analyte The capture agent of member.
In some embodiments, compound includes solid support, the first analyte, the second analyte, third analysis Object, the first bridge-jointing unit, the second bridge-jointing unit and detecting signal unit, wherein solid support, the first analyte, the second analysis Object, third analyte, the first bridge-jointing unit, the second bridge-jointing unit and detecting signal unit combine directly or indirectly to one another. In some embodiments, solid support is incorporated into the first analyte, and the first bridge-jointing unit is incorporated into the first analyte and second Analyte, the second bridge-jointing unit is incorporated into the second analyte and third analyte, and detecting signal unit is incorporated into third point Analyse object.In some embodiments, solid support includes the first capture agent, and it is single that the first analyte includes the first interaction Member and the second interaction unit, the second analyte include the first interaction unit and the second interaction unit, third point It analyses object and includes the first interaction unit and the second interaction unit, the first bridge-jointing unit is independently tied comprising one or more The capture agent of first interaction unit of the second interaction unit and the second analyte together in the first analyte, second Bridge-jointing unit, which includes, one or more is independently incorporated into the second interaction units of the second analyte and third analyte The capture agent of first interaction unit, and detecting signal unit includes the second interaction for being incorporated into third analyte The capture agent of unit.
In some embodiments, presently described method can be used for detecting a variety of food-bornes by using single signal Substance analyte detects food-borne causal agent.For example, sample can through handle with detach analyte (for example, can detach or Expansion of antigen or toxin or food-borne causal agent nucleic acid).Multiple analytes can be concurrently detected with approach described herein (for example, food borne pathogens body protein and/or amplicon).Method can then provide to test specificity larger confidence simultaneously And avoid false negative result.In some embodiments, indicating the existing positive findings of food-borne causal agent needs detection 2,3 Or 4 kinds of analytes.The method of the present invention can be used for concurrently testing and analyzing object with single signal.Single signal provides simpler As a result it explains, because signal can only will be detected when the multiple analytes detected are present in sample.Cause This, if detecting 2 kinds of analytes, then signal can only will detect in the presence of two kinds of analytes are equal.In some realities It applies in mode, signal can only detect in the presence of 3 kinds of analytes.Such method can be applied to other detection sides Method.
In some embodiments, method can be used for detecting 3 alanysis objects to provide the positive survey about food pollution Examination.In some embodiments, one kind in analyte is toxin (for example, shiga toxin 1 and/or 2).Toxin sheet can be detected Body can detect coding or control the nucleotide sequence of the generation of toxin.In some embodiments, one kind in analyte It is eae genes, is referred to as virulence factor.Eae genes can be found in or be expressed in such as enterohemorrhagic escherichia coli.
In some embodiments, one kind in analyte is serotype analyte, can be by food-borne causal agent Strain specificity generate antigen.In some embodiments, serotype analyte is e. coli serotype.In some realities It applies in mode, e. coli serotype O26, O45, O103, O111, O121 and O145.Therefore, in some embodiments, Positive test about food source contact scar needs to detect 3 kinds of analytes with single signal, wherein 3 kinds of analytes are shiga toxins (for example, shiga toxin 1 and/or 2), eae genes and the serotype analyte selected from e. coli serotype, serotype analyte It is O26, O45, O103, O111, O121 and O145.In some embodiments, serotype analyte is by pathogenic strains spy The protein of opposite sex expression.In some embodiments, analyte is the nucleic acid sequence of coding for antigens.In some embodiments In, nucleic acid sequence is the segment of the coded sequence of antigen.Specific fragment is not crucial and those skilled in the art Sequence or its segment can be determined so that conventional method to be used to be expanded.As discussed herein, it can expand and optionally use Heterologous interaction unit label target sequence.Can then according to provided herein is method test and analyze object.
For example, if the positive test about virus is needed, there are two different nucleic acid sequences, then the two nucleic acid Sequence can concurrently be detected using approach described herein with single signal, such as with detecting this respectively with more than a kind of signal Two nucleic acid sequences compare.In addition, if the presence of cancer needs to detect the several genes expressed in sample, then Ke Yitong It crosses and expresses relevant analyte (for example, amplification gene product is come by using RT-PCR) according to described herein using with it Method concurrently detects gene with single signal.Therefore, presently described method has broad applicability and can be with multiple point It analyses object (target molecule) and is even used together with different analyte.
In some embodiments, the method for detecting two or more analyte is provided, is flowed (vertically comprising using Or laterally) device detection multiple analytes.The example of vertical current device and its application method are provided in United States Patent (USP) US 8,012, 770th, US 8,183,059 and U.S. patent application case US13/500,997, US 13/360,528, in US 13/445,233, respectively Case is incorporated by herein by reference.Device can be adjusted for detecting multiple analytes using single signal.
Therefore, embodiment provided herein is provided by using vertical current and the single letter of device using vertical current Number detection multiple analytes method.Vertical current allows analyte and/or sample flow to cross layer/film of analysis analyte detection membranous system. " passing through layer " or " passing through film " is intended to refer to sample and flows through layer and vertically through the layer.In some embodiments, sample It will not flatly or laterally flow through different layers/film.
Following term is used in combination with the description of various vertical current devices.Also illustrate in the whole text with some vertical current devices or Other related terms of its purposes.
Term " pressure actuator " and " power actuator " are used interchangeably and refer to can be for example by applying force to implement The component of pressure.Power actuator is referred to as force member.Example includes but is not limited to various reinforcings described herein Component.Other examples include but is not limited to piston or other solid support structures.Power actuator relative to another component position Putting can improve, reduce or transverse shifting.Control force actuator can be carried out manually or by signal processing unit (for example, computer) Position.The ability of the position of control force actuator can be used for adjustment and be applied to another component and (such as (but not limited to) analyze Analyte detection membranous system) power (such as pressure).By adjusting the power for being applied to membranous system, the flow rate of sample can be adjusted. Power may be used to that sample is kept constant by the flow rate of membranous system or flow rate can be variable.Flow rate It can also stop and sample is made to rest on the different layers of membranous system.For example, when sample contacts bonding pad, the flowing speed of sample Rate can be zero or near zero.After being held on bonding pad, it can be increased by adjusting the pressure applied by power actuator Flow rate.Sample by entire membranous system or then can adjust applied power so that sample to be allowed to stop and (shelve) On another layer of membranous system.It, can be manually or by using signal processing unit (example when samples vertical flowing through membranous system Such as, computer) power is accurately adjusted, flow rate can be changed at any time.Flow rate can also be based in membranous system The absorbability of film and/or the number of the film of system adjust.Based on absorbability, (for example, increasing or decreasing) flowing can be adjusted Rate.
Flow rate can be measured with any unit for l/ seconds including but not limited to μ l/ minutes or μ etc..Retention period Flow rate can be such as 0 μ l/ seconds or less than 1,0.9,0.8,0.7,0.6,0.5,0.4,0.3,0.2 or 0.1 μ l/ seconds or μ L/ minutes.Flow rate can be monitored and equally be adjusted manually or by signal processing unit (for example, computer). In addition to approach described herein, method well-known and conventional known to those skilled in the art can also be passed through To adjust and monitor flow rate.In some embodiments, flow rate be about 0 Dao 1ml/ minutes, about 0-10ml/ minutes, About 1-9ml/ minutes, about 1-8ml/ minutes, about 1-7ml/ minutes, about 1-6ml/ minutes, about 1-5ml/ minutes, about 1-4ml/ points Clock, about 1-3ml/ minutes, about 1-2ml/ minutes, about 0.5-1.5ml/ minutes, about 1-1.5ml/ minutes or about 0.5-1ml/ minutes. In some embodiments, flow rate is about 1,2,3,4,5,6,7,8,9 or 10ml/ minutes.In some embodiments, it flows Dynamic rate is at least 1,2,3,4,5,6,7,8,9 or 10ml/ minutes.In some embodiments, flow rate 1,2,3,4, 5th, 6,7,8,9 or 10ml/ minutes.
In some embodiments, for including shell with the device of single signal detection multiple analytes, shell includes First casing component and second housing component.In some embodiments, the first and second casing components can be constructed as single Unit.Shell can include import.Import allows to introduce the sample into chromatography.In some embodiments, the first shell Component includes import.Import, which can have, to be enough to dispose the size for the appropriate volume for being added to the solution in device.In some realities Apply in mode, opening (opening) size be large enough to disposition about 0.1 to 3ml, about 0.1 to 2.5ml, about 0.5 to 2.0ml, About 0.1 to 1.0ml, about 0.5 to 1.5ml, 0.5 to 1.0ml and 1.0 arrives 2.0ml.
In some embodiments, shell includes bonding pad, permeable membrane, test film and/or absorption component.In some implementations In mode, shell includes analysis analyte detection membranous system.In some embodiments, analysis analyte detection membranous system include bonding pad, Permeable membrane, test film and absorption component.In some embodiments, analysis analyte detection membranous system is free of permeable membrane.In some realities It applies in mode, analysis analyte detection membranous system includes in the following order：Bonding pad, permeable membrane, test film and absorption component.
As used herein, term " bonding pad " refers to that the film of capture agent or other kinds of material can be included.With reference to Pad can be cellulose acetate, nitrocellulose, polyamide, makrolon, glass fibre, film, polyether sulfone, regenerated cellulose (RC), polytetrafluoroethylene (PTFE) (PTFE), polyester (such as polyethylene terephthalate), makrolon (such as 4,4- hydroxyls-hexichol Base -2,2'- propane), aluminium oxide, mixed cellulose ester (such as mixture of cellulose acetate and nitrocellulose), nylon (example Such as polyamide, hexa-methylene-diamines and nylon66 fiber), polypropylene, PVDF, high density polyethylene (HDPE) (HDPE)+nucleating agent " hexichol first Sour aluminium " (DBS) (such as 0.024 HDPE DBS (Porex) of 80u) and HDPE.The example of bonding pad also includes (polyethylene terephthalate),(polyethylene terephthalate),(acetic acid is fine Dimension element and nitrocellulose), water it is graceful (Whatman)(cellulose acetate and nitrocellulose), the graceful #12-S of water are (artificial Silk)),(aluminium oxide),(aluminium oxide), Sai Duolisi (Sartorius) (cellulose acetate, example Such as 5 μm) and the graceful standard 17 of water (silver soldering glass).Bonding pad can also be by contacting the material dissolved later with sample or other liquid Material is made.It can be combined the dissolving of pad so that other layers of system described herein can appear or expose for naked eyes Check (for example, detection and analysis object) or for spectrometer inspection (for example, passing through spectrometer analyte).
In some embodiments, bonding pad or test film include capture agent.In some embodiments, make bonding pad Or test film is contacted with capture agent and is then made it dry, and is used subsequently in vertical current device.Bonding pad or test film also may be used To preserve capture agent comprising other compositions so that it can stabilization be deposited at room temperature or under refrigeration or cryogenic temperature Storage.In some embodiments, bonding pad or test film are impregnated before application capture agent with buffer solution.In some embodiment party In formula, buffer solution is the blocking buffer solution for preventing non-specific binding.In some embodiments, buffer solution includes boric acid Salt, BSA, PVP40 and/or Tween-100 or its any mixture.In some embodiments, buffer solution for 10mM borates, 3%BSA, 1%PVP40 and 0.25%Tween-100.In some embodiments, capture agent is comprising trehalose and sugarcane Pad or film are applied in the solution of sugar.In some embodiments, capture agent is comprising trehalose, sucrose and phosphate And/or pad, film or both are applied in the solution of BSA.In some embodiments, capture agent is in 5% trehalose, 20% It is applied in the solution of sucrose, 10mM phosphate and 1%BSA.In some embodiments, test film includes and is incorporated into labeled expansion Increase the capture agent of son.In some embodiments, capture agent for identification or be incorporated into digoxin, fluorescein (such as FITC), the antibody of rhodamine (TAMRA) etc..
In some embodiments, bonding pad includes Streptavidin.Streptavidin can also as described herein into One step is labeled.In some embodiments, Streptavidin is incorporated into detect multiple analytes with single signal The capture agent of biotinylated antibody.
In some embodiments, the first surface of removable member contact bonding pad and adhesion component (adhesive Member the second surface of bonding pad) is contacted.
In some embodiments, device includes adhesion component.Adhesion component, which can include, allows sample to flow through bonding pad And the adhesion means inlet of engaged test film.In some embodiments, adhesion means inlet has with removable member entrance There are identical size or shape.In some embodiments, adhesion means inlet and removable member entrance with different sizes or Shape.In some embodiments, the entrance in adhesion component has same shape, but with different area.With different sides Long-pending entrance will be considered as having different sizes.Adhering component can be by being suitable for a kind of component or film being adhered to another component Or any substance of film is made.In some embodiments, adhesion component impenetrable liquid.In some embodiments, adhesion structure Part contacts removable member.
In some embodiments of device, permeable membrane is to be connected to or be adhered to test film.In some embodiments, Permeable membrane is laminated in test film.Permeable membrane can be any material that sample (such as fluid sample) is allowed to flow through test film Film.The example of test film includes but is not limited to nitrocellulose, cellulose, glass fibre, polyester, polypropylene, nylon etc.. In some embodiments, permeable membrane includes opening.Opening may have to allow to estimate or detect test film.In some implementations In mode, the opening in permeable membrane substantially has identical size with the import in shell.The example of permeable membrane includes (but unlimited In) Protran BA83, water be graceful etc..
As discussed herein, an example of solid support is test film.As herein and used in the whole text, " test film " is Refer to the film of the wherein binding partners of detection capture agent.Test film includes but is not limited to nitrocellulose membrane, nylon membrane, gathers partially Fluoride film, poly (ether sulfone) film etc..Test film can be that can be used to detect the combination of capture agent by those skilled in the art The existing any material of companion's (for example, labeled analyte, antigen or epitope).Test film can also include capture examination Agent.In some embodiments, test film contacts with capture agent and makes capture agent dry and be adhered to test film.Test film Example to include but is not limited to graceful Protran BA83, water, Opitran BA-SA83 and 0.22 μm of white light facial mask (close Li Bo (Millipore) product identification SA3J036107).Test film can also include the nanoparticle-based combined with capture agent Matter.Nanocrystal can be arranged to have the such as (but not limited to) particle based on carbon, gold or metal alloy particle, copolymer matrix Matter and monodisperse semiconductor, magnetism, metal and ferroelectric nano crystal the 2D thin slices of material and 3D matrix.Test film can wrap Containing a variety of capture agents.In some embodiments, test film includes 1,2,3,4,5,6,7,8,9 or 10 kind of capture agent. In some embodiments, test film includes multiple regions respectively with different capture agents.In some embodiments, Duo Gequ Domain not exclusively overlaps each other or unanimously.
In some embodiments, device or shell also include absorption component.Absorption component be referred to as " core pad " or " wicking pad ".Absorption component absorption is flowed through the fluid of device when sample administration is in device and is provided when sample administration is in device Contribute to the wicking-power of sample flow." absorption component " is intended to refer to have and draws (core) from the surface with material and keep molten The material of the ability of liquid.It can also allow sample is controlled to move using the combination for the material for increasing or decreasing absorptivity.
Absorption component can be for that can promote sample to flow through bonding pad and flow to any material of test film.The reality of absorption component Example includes but is not limited to cellulose, super-absorbent polymer, fiberglass packing (such as C083 (Mi Libo (Millipore))) Deng.In some embodiments, shell includes multiple (for example, 2 or 2 or more) absorption components.In some embodiments, Shell includes 2,3,4 or 5 absorption components.In some embodiments, device includes an absorption component.In some embodiment party In formula, absorption component includes one or more films (up to 10 indivedual films), and each film can be identical material or not Same material.In some embodiments, device is only made of 1 film as absorption component.
In some embodiments, device includes force member.The example of force member is described below and is found in In figure.These examples are force member that is unrestricted and can using other forms.In some embodiments, it reinforces Component can be used for applying the other assemblies of pressure or compression analysis analyte detection membranous system so that abutting against each other.In some embodiment party In formula, force member can be generated by any material for including but is not limited to plastics or stainless steel.As shown in Figure 23, fixture It can be used as force member.Stainless steel can be laser cutting so that it may be used as fixture.The nonrestrictive reality of these fixtures Example is found in Figure 23.Force member is used to apply pressure to membranous system.Force member is not limited to fixture, but can be any Shape (referring to the figure about non-limiting examples), can be to membranous system (for example, nanoparticle matrix) and positioned at sub-assembly Piston spline structure at interior key position applies pressure.In some embodiments, force member is piston.Force member can Other assemblies for applying pressure or compression analysis analyte detection membranous system to abut against each other.In some embodiments, Force member can include axis and head.Force member can have mushroom-shaped shape, and wherein head is wider than axis.In some embodiments In, head is narrower than axis.Force member comprising head and axis can be single unit or can form force member by being in contact with each other Multiple components are made.For example, head can be a unit that can be detached with axis.In assembling, head and axis are in contact with each other Force member is made.In another example, head and axis are a bonding elements and manufacture together rather than assemble formation later The individual components of force member.Force member allows device to work together with vertical current, such as with comparing dependent on transverse flow.
In some embodiments, the surface of force member contact absorption component.In some embodiments, force member Contact the surface of absorption component and the surface of layer can be removed.In some embodiments, force member is from the upper of film detecting system Side and lower section compressive films detecting system.For example, in some embodiments, force member can be clipped in all of film detecting system Layer is intermediate.In some embodiments, force member is connected to supporting member.
In some embodiments, device is in the following order comprising removable member, bonding pad and adhesion component.
Device can also include supporting member.In some embodiments, the surface of supporting member contact absorption component.Branch Support component can also have supporting member entrance.Entrance can have phase with the entrance in removable member and/or adhesion component Same size and/or shape.In some embodiments, supporting member includes and entering in removable member and/or adhesion component Mouth has the entrance of different size and/or shapes.Can supporting member be made by any material, including but not limited to plastics. In some embodiments, second housing component is used as supporting member.
Apparatus described herein can be used in the existing analysis for detecting the binding partners of capture agent.These analyses It can be used to detect multiple analytes for the detection of single signal as shown here.For example, can this be used by antibody Invention device detects antigen.Term " vertical current " uses in the whole text.Term " vertical current " refers to sample flow by being present in dress The direction of different films and component in putting.Vertical current refers to flow through the sample of film (for example, top-to-bottom), such as with transverse flow phase Comparison, transverse flow refer to flow through the sample of (for example, side to side) film, pad or absorption component.In cross-flow devices, Film and pad are horizontally located at position adjacent to each other, are substantially on the same plane.In vertical current device, each film or pad are basic Substantially different space plane in upper parallel or fully parallel to each other and occupying device.Film and pad can it is compressed at its or Similar plane is occupied when placing under stress.In some embodiments, at least part of each component, film or pad is being pushed up each other Portion's stratification.In some embodiments, at least part of each layer of component, film or pad is substantially parallel to each other.In some realities It applies in mode, at least part of each layer is in the space plane different from other layer.
In order to which vertical current is made effectively to occur, in some embodiments and when it is present, bonding pad, permeable membrane, test Film and absorption component are substantially parallel to each other.In some embodiments, bonding pad, permeable membrane, test film and absorption component are deposited It is in different space planes.In some embodiments, for shell also comprising hydrophobic membrane, sample can be slowed or stopped in hydrophobic membrane The vertical current of product.Hydrophobic membrane can be contacted with test film, this will make sample stop or be shelved in test film.Stop can allow Increased sensitivity and detection.Vertical current is adjusted by being applied to the pressure of film, pad and/or component.In some embodiments In, apply pressure perpendicular to test film and/or bonding pad.In some embodiments, pressure can be applied so that bonding pad supports It is compressed by shell.Compression against shell may be such that bonding pad is in direct contact or passes through intermediary with shell, o-ring or hoop Contact so that bonding pad and test film abut against each other compressed.
Force member can apply the pressure substantially perpendicular to test film.Without being bound by any particular theory, pressure promotees Into vertical current.Pressure makes each layer of membrane stack be contacted with another layer.Pressure can also solve divided by stop flowing so that test sample It can stop or be shelved in test film, this measure can allow bigger sensitivity.Pressure can then apply vertical to allow again Stream continues that sample is made to flow into one or more absorption components.Force member can apply pressure so that bonding pad contacts shell A part (for example, first or second casing component or layer can be removed).In some embodiments, when bonding pad is not reinforcing When under the pressure that component applies, bonding pad contact shell, but on stressed force member is applied, bonding pad is against the one of shell Part is compressed.
In some embodiments, the circumference of bonding pad contact import.Import can also include hoop or other are similar special Sign, such as o-ring.In some embodiments, bonding pad contact hoop and/or the circumference of o-ring.In some embodiments In, bonding pad can against import circumference it is compressed, import can include hoop and/or o-ring in some embodiments.
" can against import circumference it is compressed " refer to film or pad (for example, bonding pad) directly contacted with the circumference of import Another layer or material (for example, film) compressed or contact against the circumference with import are compressed.
In some embodiments, bonding pad is not in direct physical contact with shell, but is contacted with shell in fluid. " fluid contact " means if sample is applied to device by import or other openings, then fluid will contact bonding pad.One In a little embodiments, bonding pad can be detached by another film (such as permeable membrane) with shell, wherein another film is in straight with shell Connect physical contact or with hoop or o-ring in direct physical contact.When sample administration is in device, fluid can contact another first Film and then contact bonding pad.This is exactly in bonding pad example that fluid contacts with shell.There are other numerous realities Mode is applied, wherein bonding pad is not in direct physical contact with shell, hoop or o-ring, but is contacted with shell in fluid.
Force member, which can apply, to be enough to promote any pressure of the vertical current by different film layers.In some embodiments In, the layer (for example, bonding pad, permeable membrane, test film and absorption component) of device is arrived selected from about 5lbf to 100lbf, about 5lbf Through pressure under the power of 50lbf, about 10lbf to 401bf, about 15lbf to 40lbf, about 15lbf to 25lbf or about 30lbf to 40lbf Contracting.In some embodiments, the layer (for example, bonding pad, permeable membrane, test film and absorption component) of device is selected from about 1lbf It is arrived to 100lbf, about 1lbf to 50lbf, about 1lbf to 51bf, about 1lbf to 10lbf, about 1lbf to 15lbf, about 1lbf It is compressed under the power of 20lbf, about 1lbf to 30lbf or about 1lbf to 25lbf.If hydrophobic membrane or impermeable membrane are present in device In, power can also compress hydrophobic membrane or impermeable membrane.
In it can be used for some embodiments with the device of single signal detection multiple analytes, force member contact The first surface of absorption component.In some embodiments, bonding pad engaged test film.In some embodiments, test film First surface contact permeable membrane.In some embodiments, the second surface of the second surface contact absorption pad of test film. In some embodiments, device includes hydrophobic membrane and for example, the second surface of hydrophobic membrane engaged test film.In some embodiments In, hydrophobic membrane contacts the first surface of absorption pad.In some embodiments, bonding pad contact adhesion component.In some implementations In mode, test film contact adhesion component.
In it can be used for some embodiments with the device of single signal detection multiple analytes, the first of bonding pad Surface contacts the first surface of the second surface contact permeable membrane of shell and bonding pad, and the second surface contact of wherein permeable membrane is surveyed Try the second surface of the first surface of film, the wherein first surface, wherein absorption pad of the second surface contact absorption pad of test film Contact force member.In some embodiments, the circumference of the import of the first surface contact shell of bonding pad.In some implementations In mode, the first surface contact hoop of bonding pad or the circumference of o-ring.
In it can be used for some embodiments with the device of single signal detection multiple analytes, the first of bonding pad Surface contacts the first surface of the second surface contact adhesion component of shell and bonding pad, wherein the second surface of adhesion component connects Touch the second of the first surface of test film, the wherein first surface, wherein absorption pad of the second surface contact absorption pad of test film Surface contacts supporting member.In some embodiments, the circumference of the first surface contact entrance of bonding pad.In some embodiment party In formula, the first surface contact hoop of bonding pad or the circumference of o-ring.
Device can also include connecting elements.In some embodiments, connecting elements is flexible or by flexible material It is made.In some embodiments, connecting elements is made of fixed or by non-flexible material.Fixed connecting elements can be Such as hinge etc., can such as contact system bonding pad or another layer or film and its displacement can be mediated.Fixed company Other compressible materials that connection member, such as (but not limited to) fixed hinge or hinge-like work, when compressing release It can restore shape or size.Connecting elements can shift bonding pad.Connecting elements can moreover be only plastics and although can Bending, flexural property are not used to the function of device.
Flexible material can be such as elasticity or elastomeric material.Connecting elements can for example be connected to bonding pad and/or Hydrophobic membrane.Connecting elements is also connected to any film or component of device.The example of connecting elements includes but is not limited to bullet Property body band, rubber strip, spring etc..In some embodiments, connecting elements can be made of shape-memory material.In some realities Apply in mode, connecting elements make it possible to moving locking member connect with mobile bonding pad or with connecting elements it is any its Delay is generated between the film or pad of his type.In some embodiments, the movement of pad or film will not with mobile sliding button or Locking component occurs simultaneously.In it can be used for some embodiments with the device of single signal detection multiple analytes, and And without being bound by any particular theory, when sliding button or locking component move, energy is gathered in connecting elements and this Kind energy is connected to the pad or film of connecting elements for tensing after pressure has discharged.In some embodiments, mobile or Before removing bonding pad, locking component is far from force member movement (that is, force member no longer contacts locking component) in some realities It applies in mode, once it is to move to remove the compression applied by force member or pressure, bonding pad completely.
Connecting elements is also connected to sliding button or locking component.Connecting elements can by any mode, such as Sticker, bail, tying etc. are connected to other assemblies.In some embodiments, film or pad have recess in film or pad, Connecting elements is allowed to be connected to film or pad.Non-limiting examples are found in Fig. 9.
In it can be used for some embodiments with the device of single signal detection multiple analytes, shell includes locking Component.Locking component can be the slidably locking component that can be moved in the device.Locking component can be used for that structure will be reinforced Part is locked in a position so that the power generated on the different layers by force member is maintained.Locking component is will for example to add Power component is locked in a position so that pressure can not release, until locking component movement with allow force member change position (that is, reduction).Locking component can be for example mounted on the lower section of the head of force member, this will make force member be maintained at raising Position.Locking component can be with located so that force member is maintained at specific position (for example, improve or reduce) by it.It can Locking component is made by any material for including but is not limited to plastics etc..Locking component can for example directly or indirectly through Prevent another component touch force member that force member releases stress.In some embodiments, locking component contact reinforcing Component is to compress bonding pad.
Locking component can also connect component so that the movement of locking component by mobile connection component, be connected to Any other film (for example, bonding pad, hydrophobic membrane, test film or absorption component) or other assemblies of connection member.If for example, lock Component movement is determined to release the pressure of force member, and force member is thus made to change position (for example, from the position mentioned to reduction Position), then the movement of locking component also deform/will accumulates to energy in connecting elements connecting elements, therefore once Pressure fully reduces, locking component moveable diaphragm or pad.When bonding pad is connected to connecting elements and locking component moves, one Denier pressure fully reduces, and this measure will also mobile bonding pad.In some embodiments, pressure is completely removed.Bonding pad can For example to move so that it is removed from device.In some embodiments, bonding pad movement by import to appear test film. The amount of the test film appeared will be depending on the type of detection used.Naked eyes are detected, can need to appear more in import More test films.Non- naked eyes (for example, fluorescence, near-infrared, infrared, radioactivity or chemiluminescence) are detected, can need to show Reveal fewer or more test films.In some embodiments, bonding pad moves so that no longer can see that or examines by import Measure bonding pad.In some embodiments, the movement of bonding pad can generate another opening in addition to import to estimate or Detect test film.In some embodiments, bonding pad dissolves to estimate or detect test film (for example, being detected with single signal Analyte or multiple analytes).Bonding pad can be made of soluble material so that when bonding pad and sample or another solution connect When touching, bonding pad partially or completely dissolves.
In it can be used for some embodiments with the device of single signal detection multiple analytes, connecting elements also connects It is connected to impermeable membrane or hydrophobic membrane.It is mobile also by mobile or removal impermeable membrane or hydrophobic membrane when connecting elements moves.Such as this paper institutes It discusses, impermeable or hydrophobic membrane presence can make test sample stop or be shelved on test film by being slowed or shut off vertical current On.When impermeable membrane or hydrophobic membrane are mobile or remove, by the way that it is made to be connected to connecting elements or by other means, vertical current is not It is prevented from again or inhibits.
In it can be used for some embodiments with the device of single signal detection multiple analytes, shell, which includes, to be slided Button.Sliding button is alternatively referred to as sliding component.Sliding button can pass crosswise the inner surface and appearance of (cross) shell Face.In some embodiments, sliding button or sliding component are projected into the outer surface of shell.In some embodiments, it is sliding Dynamic button is directly or indirectly connected to locking component.When sliding button connection (direct or indirect) is in locking component, slip is pressed The movement of button also moving locking member.Connecting elements can be connected to sliding button in some embodiments.In some implementations In mode, connecting elements is connected to sliding button and locking component.Sliding button and locking component are also configured such that single unit.
Can be used for single signal detection multiple analytes device some embodiments in, any one or it is more A entrance (inlet) is included selected from about 0.2 to about 20cm2Range opening (opening).In some embodiments, Any one or more entrances are about 1 diameter for arriving about 2cm.In some embodiments, any one or more entrances are equal It is the diameter of about 1 or about 1.5cm.In some embodiments, any one or more entrances be about 1, about 2, about 3, about 4 or The diameter of about 5cm.In some embodiments, there is more than one entrance, entrance can be different sizes or identical size.Respectively The size of entrance is independent of one another.In some of apparatus described herein and system embodiments, device or system include 1, 2nd, 3,4 or 5 entrances.In some of apparatus described herein and system embodiments, device or system include at least 1, 2nd, 3,4 or 5 entrances.
In it can be used for some embodiments with the device of single signal detection multiple analytes, import is included and is selected from About 0.2-20cm2Range opening.In some embodiments, import is about 1 diameter for arriving about 2cm.In some embodiment party In formula, import is the diameter of about 1 or about 1.5cm.In some embodiments, import is about 1, about 2, about 3, about 4 or about 5cm Diameter.
In it can be used for some embodiments with the device of single signal detection multiple analytes, for testing and analyzing The device of object includes first component and second component.In some embodiments, first component and second component are in contact with each other. In some embodiments, first component includes one or more entrances.In some embodiments, between the first and second components To analyze analyte detection membranous system.In some embodiments, the analysis analyte detection membranous system between the first and second components includes Bonding pad, adhesion component, test film and absorption component.In some embodiments, analyte detection membranous system is analyzed in the following order Comprising：Bonding pad, adhesion component, test film and absorption component.As discussed herein, in some embodiments, bonding pad, survey At least part in examination film and absorption component each is substantially parallel to each other.In some embodiments, bonding pad, test At least part in film and absorption component each is in different spaces plane.
In it can be used for some embodiments with the device of single signal detection multiple analytes, analyte detection film is analyzed System is compressed between (for example, force member) first and second components.In some embodiments, analyte detection film is analyzed System is compressed between the plane formed by first component and the plane formed by second component, wherein by the first and second structures Part formed plane be substantially parallel to each other and with analysis analyte detection membranous system it is parallel.In some embodiments, plane is each other It is parallel and parallel with analysis analyte detection membranous system.In some embodiments, the first and the of compression analysis analyte detection membranous system Two components are force members.For example, force member is properly termed as comprising the first and second components generating compression analysis analyte detection The power of membranous system.
In it can be used for some embodiments with the device of single signal detection multiple analytes, the first and second structures Part is connected to each other along the edge of the first component at the edge for being parallel to second component.In some embodiments, first and second Component is by connections such as spring, hinges.The mode of first and second components connection is unrestricted and can be by making score Analyse object membranous system can between the first and second components compressed any structure.In some embodiments, first and Two components are adjacent to each other and formation fixture.The example of fixture (for example, force member) present application in the whole text it is middle display (example Such as Figure 16).Fixture for example from metal or can make first component have flexible any kind of material cutting so that analyte Detection membranous system may be inserted between the first and second components.In some embodiments, first component is removable.
In it can be used for some embodiments with the device of single signal detection multiple analytes, first component and knot The connection of conjunction pad or contact, the wherein movement of first component or removal movement bonding pad remove bonding pad from device.In some realities It applies in mode, bonding pad is removable.
In it can be used for some embodiments with the device of single signal detection multiple analytes, bonding pad is to pass through It only removes bonding pad and is removed from the device comprising the first and second components.
In it can be used for some embodiments with the device of single signal detection multiple analytes, bonding pad, which includes, to be adjusted Full wafer (tab).Trimmer can be used for removing or promote to remove bonding pad.
It is described herein in it can be used for some embodiments with the device of single signal detection multiple analytes Device is positioned in container.In some embodiments, container is capsule or bag.In some embodiments, container includes entrance. In some embodiments, container include can be removed or movable member or layer, component or layer exposed in movement or removal into Mouthful, allow sample administration in analysis analyte detection membranous system.It can be removed or the example of movable member or layer includes but is not limited to Wing flap (flap) or trimmer.Wing flap or trimmer are for example shown in Figure 18 and Figure 19.In some embodiments, it can be removed Layer or displaceable layers are also used as the sealing (seal) of container.Sealing can protect bonding pad and/or analysis analyte detection membrane system System.
In some of apparatus described herein and system embodiments, it can be removed or displaceable layers are contacted with bonding pad Or connection.
In it can be used for some embodiments with the device of single signal detection multiple analytes, for testing and analyzing The device of object includes the first external component (outer member) and includes the first internal component (inner member) and second Second external component of internal component, wherein the first internal component and the second internal component are in contact with each other.In some implementations In mode, the first external component includes entrance.In some embodiments, the first internal component includes entrance.In some implementations In mode, the first external component and the first internal component include entrance.In some embodiments, in the first and second inside structures It is analysis analyte detection membranous system between part.In some embodiments, device includes bonding pad.In some embodiments, it fills Put shortage bonding pad.In some embodiments, analysis analyte detection membranous system includes test film and absorption component and optionally Include bonding pad.In some embodiments, analysis analyte detection membranous system includes test film and absorption component in the following order. In some embodiments, at least part in optional bonding pad, test film and absorption component each is substantially put down each other Row.In some embodiments, as discussed above, analysis analyte detection membranous system is in the first internal component and the second inside structure It is compressed between part.In some embodiments, device and/or system include adhesion component as described herein.At some In embodiment, device includes filter membrane.In some embodiments, filter membrane can be in analysis analyte detection membranous system.At some In embodiment, the first surface of filter membrane contacts the surface of the first internal component and the second surface contact analysis quality testing of filter membrane Survey another film or component of membranous system.In some embodiments, the surface of the second surface engaged test film of filter membrane.Filter membrane can Think any material as described herein.For example, in some embodiments, filter membrane can be that can be used as bonding pad, test The identical material of film, absorption component etc..In some embodiments, filter membrane is fiberglass packing.
In it can be used for some embodiments with the device of single signal detection multiple analytes, when bonding pad is not deposited When being in device or system, conjugate is in liquid form or in being dissolvable in water liquid (for example, water, buffer solution, physiology salt Water etc.) in material forms supply.Conjugate can be supplied and in autonomous container (for example, pipe) by dress described herein It puts or system provides.Conjugate be in a reservoir for seasonable, conjugate be sample administration in analysis analyte detection membranous system it It is preceding to be incubated with (incubate) with sample.Sample can be generated by any method and/or as described herein.For example, one Block meat can be through cleaning or wiping and generate test sample.Test sample then can be incubated with or contact with conjugate to generate Test sample-conjugate mixture.This mixture then can apply application using device as described herein and/or system In analysis analyte detection membranous system as described herein.In some embodiments, test sample-conjugate mixture is directly applied For test film.In some embodiments, test sample-conjugate mixture is filtered before engaged test film or wears Cross another film.
In it can be used for some embodiments with the device of single signal detection multiple analytes, analyte detection film is analyzed System is compressed between the first and second internal components.In some embodiments, analysis analyte detection membranous system is by first It is compressed between plane that internal component is formed and the plane formed by the second internal component, wherein by the first internal component and the Two internal components formed plane be substantially parallel to each other and with analysis analyte detection membranous system it is parallel.In some embodiments In, plane is parallel to each other and parallel with analysis analyte detection membranous system.In some embodiments, plane is arranged essentially parallel to One and second external component.
Herein, in some embodiments of described device in the whole text, bonding pad is not by the first and second inside structures Part is compressed by force member described herein.
In it can be used for some embodiments with the device of single signal detection multiple analytes, the first external component Include removable or removable trimmer.In some embodiments, bonding pad is to be connected to first external component.One In a little embodiments, bonding pad is to be connected to removable or removable trimmer.In some embodiments, the first external component Container is formed with the second external component and container encapsulates first and internal second component.In some embodiments, container be capsule, Bag is (for example, sealable (such as slide fastener, adhesion etc.) or can surrounding for any other type analyze analyte detection membranous system and pressure The container to contract between the first and second internal components.
In it can be used for some embodiments with the device of single signal detection multiple analytes, container includes removable Remove or move trimmer.Can be removed or removable trimmer can be any shape and can be fully can be removed or The removed degree to exposure entrance.In some embodiments, trimmer in movement or removal removes or moves bonding pad. Such as sufficient distance can be removed in bonding pad so that can analyze the result of (for example, range estimation) test film.
In it can be used for some embodiments with the device of single signal detection multiple analytes, the first of bonding pad Surface is contacted with the first external component and the second surface of bonding pad is contacted with the first internal component.
In it can be used for some embodiments with the device of single signal detection multiple analytes, in first and second Portion's member parallel is connected to each other in the edge of first internal component at the edge of the second internal component.In some embodiments In, the first and second internal components are connected by spring, hinge etc..The mode of first and second internal components connection is unrestricted simultaneously And it compressed any structure can be carried out between the first and second components by analyte membranous system.At some In embodiment, the first and second internal components are adjacent to each other and formation such as fixture.The example of fixture is shown in this Application case in the whole text in.Fixture for example from metal or can make the first internal component have flexible any kind of material cutting, So that analysis analyte detection membranous system may be inserted between the first and second components.In some embodiments, the first internal component It is removable.
As discussed herein, device and system can include removable or displaceable layers (for example, trimmer).It can be by manual Power such as (but not limited to) removes (pealing) or tear to remove or move removable or displaceable layers.It can also be by machinery Power removes or mobile removable or displaceable layers.It can be removed or the mode of displaceable layers movement can be any mode.It can be removed Or the example of displaceable layers includes but is not limited to trimmer, wing flap etc..As discussed herein, this wing flap or trimmer can be with As sealing etc..
As discussed herein, bonding pad can include analyte specificity capture agent.In some embodiments, with reference to Pad includes multiple analytes specificity capture agent.In some embodiments, bonding pad includes 1,2,3,4 or 5 kind of analyte Specific capture agent.This analyte can be can be by any molecule of capture agent specific recognition.The example of analyte Including polynucleotide molecule (such as DNA, RNA, siRNA, antisense oligonucleotides), peptide, protein, sugar, polysaccharide, carbohydrate Deng.Antigen can also refer to the different epitopes being present on same protein or polypeptide.Analyte can also refer to from pathogenicity or The antigen of avirulence organism.
In it can be used for some embodiments with the device of single signal detection multiple analytes, device can be independent Ground, in pairs or in various configurations casing.Shell can be watertight with prevent leakage and can by a variety of inert materials, such as Polymer material manufactures.In some embodiments, entrance, which can have, is enough to accommodate any the desired amount of treat and the present invention one Act the volume of the sample used or reagent.
Because the film of device, component or pad are chemically inert in some embodiments, may must need Fix for solvent transport specificity combinating reagent activated at required any reaction site.According to the specific of reagent Chemical property may need a variety of methods to make Immobilized reagents.In general, when medium is that nitrocellulose or mixing nitrification are fine During dimension element ester, not needing to special chemical key even makes Immobilized reagents.Multiple technologies can be used for other materials and reagent, packet It includes with such as material of carbonyl dimidazoles, glutaraldehyde or succinic acid functionalization or the material processing with such as cyanogen bromide.Other are closed Suitable reaction includes with schiff bases and boron hydride processing restoring aldehyde, carbonyl and amino.DNA, RNA and certain antigens can be directed to Solvent transport on chromatographic material by baking and immobilization.Baking can be in the temperature in the range of about 60 DEG C to about 120 DEG C Degree is lower to carry out, and continues the time changed between about 5 minutes to about 12 hours, and in some embodiments, at about 80 DEG C Under carry out about 2 hours.
Embodiments described herein also provides the system comprising apparatus described herein and buffer container.System It can be used for detecting multiple analytes with single signal.Buffer container can be for that with the sample tested can mix and then apply Any buffer solution for device.For example, sample can be obtained from source and sample can be mixed with buffer solution.Buffer solution can be with For that will dissolve the dissolving buffer solution (lysis buffer, lysis buffer) of (cracking, lyse) cell or the pH of sample is maintained to make Obtain the buffer solution that can be suitably analyzed.Buffer container can be any shape and may include shell in device Outside or inside.
In some embodiments, the system comprising sample divider is provided.Sample divider can be can be from source Obtain sample and any material that sample is allowed to be tested.For example, sample divider can be swab, such as cotton swab. In some embodiments, sample divider is inoculator.In some embodiments, shell includes sample divider and sample A part for collector is in the inside of shell.In some embodiments, sample divider partly in the outside of shell and Partly in the inside of shell.In some embodiments, sample divider is fully in the outside of shell.
Also offer detects the kit of multiple analytes with single signal, and wherein kit includes dress described herein It puts.Kit can include device as described herein, sample divider, buffer container, specification, positive control, Negative control object or any combination thereof.About kit, positive control is known containing can be with being present in kit The sample of one or more analytes of device detection.In contrast, negative control object by without can by kit detect Analyte.For example, negative control object will confirm that device suitably works when being used in combination with antiantibody.
Buffer solution can also be included in the present invention.The example of buffer solution includes but is not limited to 1X PBS (10mM phosphoric acid Salt, 137mM sodium chloride, 2.7mM potassium chloride), washing buffer (for example, 10mM sodium phosphates, 150mM NaCl, 0.5% tween- 20th, 0.05% sodium azide), film buffer solution is (for example, 10mM sodium phosphates, 0.1% sucrose, 0.1%BSA, 0.2%PVP-40 PH7.21 is filtered with 0.2 μm of filter), polyclonal conjugate block buffer solution (for example, 50mM borates, 10%BSA, pH8.93)；Polyclonal conjugate diluent (for example, 50mM borates, 1%BSA, pH9.09) or block buffer solution (for example, 10mM sodium phosphates, 0.1% sucrose, 0.025%Silwet pH7.42；10mM sodium phosphates, 1% sucrose, 1% trehalose, 0.01% BSA, 0.025% Tween-20；0.05% sodium azide, 0.025%Silwet pH7.4；10mM sodium phosphates, 0.1% sucrose, 0.1%BSA, 0.2%PVP-40 pH7.21).Buffer solution can also be that (but not limited to) blocks buffer solution (for example, in deionization 10%BSA (pH7.4) in water or 1%BSA (pH7.4) in deionized water)；10mM borates, 3%BSA, 1%PVP40 With 0.25% tween -100 etc..
Bonding pad and test film can connect in capture agent presence or absence of under with any buffer solution described herein It touches and in some embodiments, makes it dry.
The example of buffer solution as dissolving buffer solution includes such as (but not limited to) 2% tween (v/v) and 0.1% Triton(v/v)；2% tween (v/v) and 0.1%SDS (w/v)；2% tween (v/v) and 0.1%BSA (w/v)；2% tween (v/v) and 1%BSA (w/v)；0.1%SDS (w/v), 1%BSA (w/v).Dissolving buffer solution can be for Such as 5% tween/PBS；2% tween/PBS+0.1%SDS；2% tween/PBS+1%BSA.Dissolve other example packets of buffer solution Include 5% Tween-80 of (but not limited to) (v/v)；5%Triton X-100 (v/v)；5%NP40 (v/v)；2% Tween-80 (v/ v)；2%Triton X-100 (v/v)；2%NP40 (v/v)；1% Tween-80 (v/v)；1%Triton X-100 (v/v)；With 1%NP40 (v/v).The other components of detergent (detergent) and buffer solution can be by being suitable for any suitable buffer of protein Preparation and including but not limited to water and phosphate buffered saline (PBS).Dissolving buffer solution can be in sample and dress described herein Sample is used to prepare before putting contact.In some embodiments, without using dissolving buffer solution.When it is desirable that being detected in method When surface protein or surface analysis object, without using dissolving buffer solution on sample.Therefore, in some embodiments, sample is not It is subjected to dissolving or cytolytic condition will be caused.When using cell, cell can be a part for bridge joint compound and replace Change the amplicon being for example shown in Fig. 3.Cell can be labeled or not labeled, simply by the presence of can generate similar bridge Connect the capture agent of compound.
The method that present subject matter also provides detection multiple analytes, including making sample and device as described herein And/or system contact, wherein sample contact bonding pad and test film, wherein the positive reaction with test film indicates multiple analytes Presence.In some embodiments, bonding pad includes detecting signal unit or is incorporated into the capture agent of detecting signal unit. In some embodiments, test film includes the second analyte specificity capture agent.This capture agent can be incorporated into and deposit The interaction unit being in analyte.Sample can have the expansion of such as difference (difference, differentially) label Increase son.For example, test film can include the first capture agent for being incorporated into interaction unit existing in the first analyte. Bonding pad can have the capture agent for being incorporated into detecting signal unit.It is being applied to device and is contacting bonding pad and/or test Before film, analyte can be incubated with bridge-jointing unit and/or detecting signal unit.When analyte, capture agent and signal are examined It surveys in the presence of the compound of unit, indicates positive reaction.Otherwise, signal is not generated.Capture agent can be applied to test film, make When proper its is incorporated into its specific binding partner, positive reaction will be indicated.System and device can be utilized to be formed herein Described compound.For example, after occurring to generate the PCR reactions of the amplified production of difference label, product is produced with can be used for The antibody of raw bridge joint compound is incubated with.Mixtures incubated is subsequently added in device.Sample is flowed through containing capture agent Bonding pad and then with the test membrane interaction containing another capture agent.In some embodiments, bonding pad is moved It removes or moves so that can detect signal.Movement of the bonding pad described herein in vertical current device.If all analyses Object exists, then generates bridge joint compound and can detect single signal.It can apply in test film in any way Specific capture agent causes when detection reagent, can form line, circle, plus sige, dotted line, " X " or any other pattern. In some embodiments, the control line that instruction device suitably works will cross over analyte specificity line and when a variety of analyses When object exists and is detected, detectable label will form plus sige.It can be by detecting detection reagent as described herein and leading to Conventional method known to those skilled in the art is crossed to determine detection.Similar approach can be used in such as ELISA systems.
In some embodiments, sample contact device, then after sample has contacted device, buffer solution is applied In device.For example, the sample comprising analyte can be contacted with bonding pad so that sample is transferred to bonding pad.It is connect with bonding pad After touching, another solution can be applied to device and flow vertically through apparatus described herein to promote or start.
In some embodiments, as described herein, capture agent is antibody.In some embodiments, it is tested Sample for solution, but may be solution or buffer solution and the mixture of the solid material of device can be applied to.Solution will It then dissolves one or more analytes and the capture agent of bonding pad is allowed to be connect with the appropriate analyte being present in sample It touches.In some embodiments, sample includes cell lysates.In some embodiments, cell lysates have passed through Centrifugation or other modes obtain clarifying to remove insoluble material.
In some embodiments, method includes test sample is made to contact with sample divider and makes sample divider and dress Put contact.Test sample can be the sample for including the amplicon generated by multiple analytes.In some embodiments, method Including making sample divider and solution or Buffer fluid contacts, wherein solution or buffer solution is applied to device.In some embodiment party In formula, before sample is contacted with test film, sample is contacted with bonding pad.In some embodiments, sample and bonding pad and Test film contacts simultaneously.
In some embodiments, method includes moving the bonding pad of apparatus described herein, the wherein shifting of device Dynamic exposure test film is for detection.In some embodiments, locking component movement bonding pad.In some embodiments, it ties It closes pad and is connected to locking component and/or sliding button component.In some embodiments, the movement or removal of removable member can move Dynamic or removal bonding pad.In some embodiments, bonding pad is connected to removable member and/or adhesion component.In some realities It applies in mode, when removable member is mobile or removes, adhesion component is also removed about the movement of its initial position or from device.Point It can be analyte discussed herein or any other point that methods and apparatus described herein can be used to detect to analyse object Analyse object.In some embodiments, method includes sample administration to device and sample is allowed to flow through device via vertical current.
In some embodiments, the present or absent of multiple analytes is detected or indicated by going out in less than 60 seconds It is existing.In some embodiments, the present or absent of multiple analytes is detected or indicated by occurring in about 30 to about 60 seconds. In some embodiments, the present or absent of multiple analytes is detected or indicated by occurring in less than 2 minutes.At some In embodiment, the present or absent of multiple analytes is detected or indicated by occurring in about 30 seconds.
In some embodiments, the device with single signal detection multiple analytes is provided.In some embodiments, Device includes shell.Device can include the first casing component and second housing component to form shell.In some embodiments In, the first and second casing components are individual member.First and second casing components can be fabricated to single-piece.In some embodiment party In formula, single-piece can be separated into two casing components to allow to incorporate a material into shell (such as device).In some embodiment party In formula, device includes entrance.Entrance can be in any casing component (for example, first or second casing component).Entrance can be with It is oriented to above bonding pad so that contacting bonding pad before engaged test film by the sample in entrance introducing device.Device passes through For orientation so that no matter any pressure is applied to device, sample will flow through film (for example, analysis analyte detection membranous system) vertically downward Layer.Therefore, in some embodiments, second housing component includes import.In some embodiments, second housing component It is at the top of the first casing component.Entrance can be any size as described herein or shape, as long as size and shape are sufficient To introduce the sample into device so that sample can be with contact analysis analyte detection membranous system.
Device can include one or more force members.Force member can apply pressure to analysis analyte detection membranous system Power.Power is perpendicular to or applies substantially perpendicular to the film or layer of analysis analyte detection membranous system.In some embodiments, device Include at least 1,2,3,4 or 5 force member.In some embodiments, device includes at least 1,2,3,4 or 5 reinforcing structure Part.In some embodiments, device includes multiple force members.Force member can be contacted with casing component.In some realities It applies in mode, the first surface of force member is contacted with casing component (for example, first or second casing component).In some implementations In mode, the second surface contact analysis analyte detection membranous system of force member.As described herein, force member can be used for pressing Division analyses analyte detection membranous system so that sample flow to be promoted to cross analysis analyte detection membranous system.Pressure flows through point in which can promote samples vertical Analyse analyte detection membranous system.Force member can be oriented in device independently of one another.Force member can also be through manipulating so that each It is different that force member, which applies pressure to different analysis analyte detection membranous systems and is applied to each power for analyzing analyte detection membranous system, Or be identical or substantially the same in some embodiments.
In it can be used for some embodiments with the device of single signal detection multiple analytes, device includes one Or multiple removable locking components.In some embodiments, locking component contact force member is moved.In some embodiment party In formula, removable locking component contact is present in each force member in device.For example, including the first and second reinforcing structures In the device of part, removable locking component is contacted with the first force member and the second force member.In some embodiments, may be used Moving locking member support force member so that force member is in the position of raising.Can by comparing when force member with The position of force member determines during removable locking component contact and when force member is not contacted with removable locking component The position of raising.There is no when contacting between force member and removable locking component, force member is in first position.When can When moving locking member is contacted with force member, force member is in the second position.In some embodiments, the of force member Two positions are considered as the position improved.The position of raising can be used for the layer (for example, film) of compression analysis analyte detection membranous system.When When removable locking component is not contacted with force member, in some embodiments, analysis analyte detection membranous system is not compressed.
Device can include one or more removable locking components.In some embodiments, device include it is multiple or 1st, 2,3,4 or 5 removable locking components.In some embodiments, device includes at least 1,2,3,4 or 5 removable lock Determine component.In some embodiments, the number of removable locking component that device includes is equal to the reinforcing being present in device The number of component.
Removable locking component can also include mobile member, such as (but not limited to) handle.Mobile member can be used for Such as rotation or mobile removable locking component cause locking component to contact force member.In some embodiments, mobile structure Locking component is made to be detached from from force member for part so that force member changes position (for example, from the position mentioned to the position of reduction It puts).Mobile member can be used for releasing the pressure applied on analysis analyte detection membranous system or apply the pressure.Mobile member It can be also used for releasing or apply the compression of analysis analyte detection membranous system.In some embodiments, mobile member makes locking structure Part surrounds the center axis rotation of device.For example, sample administration is flowed through at least one analysis analyte detection in device and sample After membranous system, mobile member is moved, and removable locking component is made to rotate in the clockwise or counterclockwise direction.It is removable The rotation of locking component makes force member be reduced to different positions.The rotation of removable locking component, which also allows to release, to be applied to Analyze the pressure of analyte detection membranous system.In some embodiments, pressure is fully released or in some embodiments, Pressure only partially releases.
In it can be used for some embodiments with the device of single signal detection multiple analytes, mobile removable lock The mobile member for determining component projects through first or second casing component.In some embodiments, it is exported by mobile member It is close to (accessible) mobile member.In some embodiments, mobile member surrounds the central shaft of device when moving Rotation.In some embodiments, mobile member makes removable locking component laterally move (for example, flatly) or vertically It is dynamic.In some embodiments, locking component is moved laterally (for example, flatly) or to be vertically movable when moving.
Mobile member and removable locking component can be constructed as single-piece or be two pieces.In some embodiments, wherein Removable locking component and mobile member be independent two pieces and be configured to interact with each other so that the movement of one make it is another Person moves.For example, one in two pieces can have " male member ", male member projects from the surface and is inserted into " female component " of another In with formed interaction.
Removable locking component movement is made to can be used for mobile or removal by mobile member and be present in analysis analyte detection film Bonding pad in system.As discussed herein, bonding pad can be removed to allow to estimate or analyze test film.As used herein theory It states, fully can remove bonding pad from analysis analyte detection membranous system or is removed with being enough to allow to estimate or analyze the amount of test film Bonding pad.The analysis of test film can be based only upon visual inspection or in some embodiments, can use optical pickup Analysis test film is to determine being not present or existing for antigen in sample.
In it can be used for some embodiments with the device of single signal detection multiple analytes, device includes multiple Or two or more analysis analyte detection membranous systems.In some embodiments, device includes at least 1,2,3,4 or 5 points Analyse analyte detection membranous system.In some embodiments, device includes 1,2,3,4 or 5 analysis analyte detection membranous system.Analyze quality testing Surveying membranous system can be as described in the whole text such as herein and present application.
In it can be used for some embodiments with the device of single signal detection multiple analytes, device includes one Or multiple flexible or non-flexible connecting elements.In some embodiments, device includes multiple flexible or non-flexible connecting elements. In some embodiments, device includes at least 1,2,3,4 or 5 flexibility or non-flexible connecting elements.In some embodiments In, device includes 1,2,3,4 or 5 flexibility or non-flexible connecting elements.In some embodiments, flexible or non-flexible connection The removable locking component of component contact.In some embodiments, the removable locking structure of flexible or non-flexible connecting elements contact Part and bonding pad.Flexible or non-flexible connecting elements can be used for removing or move bonding pad far from analysis analyte detection membranous system The remainder of layer (for example, film).In some embodiments, number of flexible or non-flexible connecting elements that device includes etc. In the number of analysis analyte detection membranous system being present in device.In some embodiments, the flexible connection structure that device includes The number of part is equal to the number for being present in the force member in device.Flexible or non-flexible connecting elements can be used for knot of retracting Close pad with appear or expose test film part or all.
For example, in some embodiments, device includes three analysis analyte detection membranous systems and three force members.Have The device of the analyte detection membranous system of the analysis more than one can be used for detecting different analytes or different multiple analyte groups. In this kind of device, for example, device includes first, second, and third connecting elements.First connecting elements can be with the first analyte Detect the bonding pad of membranous system and the contact of removable locking component.In addition, in some embodiments, the second connecting elements can be with It is contacted with the bonding pad of the second analysis analyte detection membranous system and removable locking component.In some embodiments, third connects Component can analyze the bonding pad of analyte detection membranous system with third and removable locking component contacts.In some embodiments, First, second, and third connecting elements is contacted with identical removable locking component.In some embodiments, first, second With third connecting elements contacted with different removable locking components.For example, in some embodiments, first and second connect Connection member is to be contacted with identical removable locking component and third connecting elements is contacted with different removable locking components. Each connecting elements is independently contacted with the removable locking component of one or more.
In it can be used for some embodiments with the device of single signal detection multiple analytes, locking structure is moved Part includes one or more removable locking component extensions (extension).In some embodiments, locking structure is moved Part extension contacts force member.In some embodiments, the number for the removable locking component extension that device includes with The number for being present in the force member in device is identical.In some embodiments, locking component extension is moved partly It surround or surrounds force member.In some embodiments, it moves locking component extension and completely surrounds or surround reinforcing Component.The shape of removable locking component or extension can be that force member is made to be maintained at any in the position of raising Shape.In some embodiments, extension is partially or even wholly surround or surrounds the hook of force member or hook sample shape. Shape is not important, as long as shape is used as the supporter of power actuator (for example, force member).
The number of removable locking component extension can be with the force member being present in apparatus described herein Number is identical or different.In some embodiments, device includes multiple removable locking component extensions.In some embodiment party In formula, device includes at least 1,2,3,4 or 5 removable locking component extension.In some embodiments, device include 1, 2nd, 3,4 or 5 removable locking component extensions.For example, in some embodiments, device includes first, second, and third Force member connecting elements and first, second, and third removable locking component extension.In this non-limiting examples, For example, the first force member contacts the first removable locking component extension, the second removable locking of the second force member contact Extension, and third force member contact third moves locking component extension.
In it can be used for some embodiments with the device of single signal detection multiple analytes, locking structure is moved Part includes connecting elements extension, and extension can be flexible or non-flexible.In some embodiments, connecting elements extends Portion connects component.In some embodiments, connecting elements extension include connecting elements extension tubercle (node, nodule).Tubercle can be to make connecting elements securely so that connecting elements maintains it and removable locking component securely Any shape or size of contact.In some embodiments, one or more removable locking component extensions are from removable Radially the extension (for example, outward) of locking component.
The number of connecting elements extension can be with the analysis analyte detection membranous system that is present in apparatus described herein Number it is identical or different.In some embodiments, device includes multiple flexible or non-flexible connecting elements extensions.One In a little embodiments, device includes at least 1,2,3,4 or 5 flexibility or non-flexible connecting elements extension.In some embodiment party In formula, device includes 1,2,3,4 or 5 flexibility or non-flexible connecting elements extension.For example, in some embodiments, dress It puts comprising first, second, and third connecting elements and first, second, and third connecting elements extension.It is nonrestrictive at this In example, for example, the first connecting elements contacts the first connecting elements extension, the second connecting elements contacts the second connecting elements and prolongs Extending portion, and third connecting elements contact third connecting elements extension.
In some embodiments, apparatus described herein includes flexible and non-flexible connecting elements and/or component prolongs Extending portion.The present invention in the whole text in, it can be mentioned that flexible or non-flexible connecting elements or extension.An if embodiment party Formula discloses flexible member, then it will be appreciated that unless opposite instruction herein, it is non-flexible another reality otherwise also to disclose wherein component Apply mode.
In it can be used for some embodiments with the device of single signal detection multiple analytes, device includes channel System.Channel system can be used for from the import traffic sample (for example, fluid) of device to one or more be present in device A analysis analyte detection membranous system.As used herein, " channel system " refers to whole system without how many respective channel considered It is a part for system.For example, channel system can include two or more from entrance by fluid transport to analysis quality testing Survey the channel of membranous system, such as (but not limited to) capillary.In some embodiments, channel system includes one or more points Branch (for example, arm).Fluid transport can be analyzed analyte detection membranous system by one or more arms to one or more.In some embodiment party In formula, channel system includes 1,2,3,4 or 5 branch.In some embodiments, the branch in the channel system that device includes Number be equal to be present in device analysis analyte detection membranous system number.
Can be used for single signal detection multiple analytes device some embodiments in, channel system it is each Branch includes the capillary by fluid transport to analysis analyte detection membranous system from entrance.In some embodiments, each branch's packet Containing multiple capillaries.In some embodiments, each branch includes at least 1,2,3,4 or 5 capillary.In some embodiments In, channel system does not include capillary or pipe sample formation (formation), but by allowing to transport a part for sample from entrance The material of the defeated bonding pad to analyte detection system is made.In some embodiments, channel system is porous material, can For sample is transported to analysis analyte detection membranous system from entrance.In some embodiments, channel system by with bonding pad Identical material is made.In some embodiments, channel system and bonding pad are an adjacent block of material.In some embodiment party In formula, channel system includes Porex materials.These porous materials allow entrance with analysis analyte detection membranous system in fluid communication. In some embodiments, porous material includes polyethylene, polypropylene, polytetrafluoroethylene (PTFE) (PTFE), PVDF, ethyl vinyl acetate (ethyl vinyl acetate), nylon 6 (Nylon 6), thermoplastic polyurethanes (TPU), SCP etc..In some implementations In mode, bonding pad and channel system are identical material or different materials.In some embodiments, channel system does not include more Porous materials and/or capillary system.
In it can be used for some embodiments with the device of single signal detection multiple analytes, channel system contact Entrance.In some embodiments, the top of channel system contact analysis analyte detection membranous system.In some embodiments, lead to The top of road system contact bonding pad or the film at the top of bonding pad.In some embodiments, channel system contact bonding pad Edge or the film at the top of bonding pad edge.How contact analysis analyte detection membranous system is unrelated with sample, in some implementations In mode, analysis analyte detection membranous system is flowed through to samples vertical.Therefore, although sample can flatly (for example, laterally) from Entrance flows to analysis analyte detection membranous system, but does not analyze sample, until it flows vertically through analysis analyte detection membranous system.This and its Middle sample laterally flows through multiple films (for example, flatly) or the lateral streaming system of test layer is significantly different.
In it can be used for some embodiments with the device of single signal detection multiple analytes, channel system is by sample Product are divided into moiety, wherein each moiety contacts independent analysis analyte detection membranous system.In some embodiments, channel Sample is divided into one or more unequal parts by system.Then transport independent analyte in one or more unequal parts Detect membranous system.
For example, in the device comprising the first and second analysis analyte detection membranous systems, device includes first and second The channel system of branch.In some embodiments, analyte detection membranous system is analyzed in the first branch contact first and the second branch connects Touch the second analysis analyte detection membranous system.When by sample administration to device (for example, via import), sample is passed through with moiety First and second branches of channel system are transported to the first and second analysis analyte detection membranous systems.In some embodiments, sample Product are transported by the first and second branches of channel system to the first and second analysis analyte detection membranous systems with unequal part.Sample Product can be divided into unequal part, such as the number based on the capillary being present in each branch.For example, the first branch can wrap Containing the capillary more than the second branch.Greater number of capillary will allow more Multi-example to pass through the first branch rather than the second branch Thus transport delivers unequal part to the first and second analysis analyte detection membranous systems.
Therefore, the branch of channel system can have equal number of capillary or different number of capillary.Channel system The number of capillary in each branch of system is independently of each branch.That is, each branch of channel system can have with another branch Same number or different number of capillary.Therefore, in some embodiments, the channel system of device can be described as capillary Tube passage system.In some embodiments, channel system is packaged in and herein with respect to device itself described first It is separated with second housing component and in different channel envelopes.In some embodiments, channel envelope is transparent, translucent , it is opaque or part translucent.
As discussed herein, can with employment eye meat eye or by machine, such as optical pickup come analyze test film with Single signal determines the existence or non-existence of multiple analytes.In some embodiments, analysis is by port (portal) It carries out.In some embodiments, device includes one or more ports, and size is enough to allow to estimate one or more analyses The test film of analyte detection membranous system.In some embodiments, each test being present in device is estimated using single-port Film.In some embodiments, device does not include port.In the embodiment for not including port in wherein device, test film is still It can be estimated by using transparent or semitransparent shell for device.In some embodiments, outside first and/or second Shell is transparent or semitransparent.When first and/or second housing are transparent or semitransparent, this can allow to analyze analyte detection film System and its test film appear in mobile or removal bonding pad.In some embodiments, device includes multiple ports. In some embodiments, device includes at least 1,2,3,4 or 5 port.In some embodiments, device includes 1,2,3,4 Or 5 ports.In some embodiments, device includes 1 port, and port is present in each in device for continuous and exposure Analyte detection membranous system is analyzed to carry out visual inspection.
As discussed herein, force member can be allowed to be moved between at least two positions (for example, improve or reduction 's；(engagement, engaged) of engagement or (unassembled, the disengaging, disengaged) do not engaged).In some embodiment party In formula, force member is to reduce and surrounded by power actuator exit.Therefore, in some embodiments, device includes one Or multiple power actuator exits, when force member reduces, export acceptable force member.In some embodiments, device Include multiple power actuator exits.In some embodiments, power actuator exit is groove.In some embodiments, power Actuator exit is circle or substantially circular.Power actuator exit may be used to power actuator (for example, force member) It is suspended on specific location.Power actuator exit can be also used for that power actuator is made to keep in the second position.In some implementations In mode, the circumference of power actuator exit is more than in force member into the circumference of the part of inlet/outlet.In some embodiments, The circumference of power actuator exit is more than the largest circumference of force member.In some embodiments, the circumference of power actuator exit No more than the largest circumference of force member, wherein force member contains the region at least two different circumference.For example, herein Described in will have there are two different circumference force member.Force member can include tool there are one the cap of circumference and with difference The support construction of the support cap of circumference.Support construction can have the circumference smaller than cap in some embodiments.In some realities It applies in mode, power actuator exit can have more than support construction circumference but less than the circumference of cap structure circumference.In some realities It applies in mode, the number of force member of the number of power actuator exit with being present in device is identical or different.
Power actuator exit can also be the continuous recess (depression) in casing component, when each in device When force member reduces and do not recompress analyte detection membranous system, outlet can receive force member.Outlet can be used for temporarily When to accommodate force member or its can be permanent so that force member can not improve to compress or further compression point again Analyse analyte detection membranous system.
As discussed in the whole text with present application herein, bonding pad, permeable membrane, test film and absorption component can by or It is being compressed under certain power of the power of about 1lbf to about 100lbf as described herein and including but not limited to by force member. In some embodiments, wherein there are multiple analysis analyte detection membranous systems, the pressure for being applied to each film detecting system can be not With or its can be identical.For example, in the device with first, second, and third analysis analyte detection membranous system, first Analyzing analyte detection membranous system can be compressed under the power of 5lbf, and the second analysis analyte detection membranous system can be under the power of 10lbf It is compressed, and third analysis analyte detection membranous system can be compressed under the power of 25lbf.In another example, implement at some In mode, the first and second analysis analyte detection membranous systems are compressed at the same pressure and third analysis analyte detection membranous system is It is compressed under the pressure different from the first and second analysis analyte detection membranous systems.The difference of pressure is used for different Flow rate, different flow rates can be adapted for different analytes.Pressure is related to flow rate.Reinforcing can be passed through The position of component manipulates pressure with the number of plies compressed in analysis analyte detection membranous system.It can be by those skilled in the art Certain force used in determining and measure using known and conventional method.
As described herein, in some embodiments, the present invention is provided comprising any device described herein, buffering The system of liquid container and/or sample divider.In some embodiments, the present invention is provided comprising any dress described herein Put and positive control, negative control object, specification, buffer container and/or sample divider or any combination thereof in One or more kits.
Approach described herein can be with analyzing analyte detection membranous systems with for example multiple (two or more) Device is used together.Method can also be used together with the device with 2,3,4 or 5 analysis analyte detection membranous systems.Exist During analyte detection membranous system (for example, 3,4,5,6,7,8,9 or 10) of the analysis more than two, change method contained herein and retouch It states with consistent with the number for analyzing analyte detection membranous system.These variations are according to description contained herein and fields Any routine changes to carry out known to technical staff.In relation to the technology based on description contained herein and fields The variation that the knowledge of personnel covers more than 2 analyte film detecting systems will not need to excessive experiment.As described herein Some embodiments in, device include two or more analysis analyte detection membranous system.In some embodiments, method Including sample (for example, sample comprising multiple analytes) is made to be contacted with device and transports a part of sample by channel system The defeated bonding pad to two or more analysis analyte detection membranous systems.In some embodiments, method includes detection and analysis The positive or negative reaction of object, wherein there are multiple analytes for positive reaction instruction.In some embodiments, by force member Compress two or more analysis analyte detection membranous systems.In some embodiments, sample vertically flows to survey from bonding pad Try film.In some embodiments, method further comprises compressing analysis analyte detection membranous system by force member.In some implementations In mode, method is included in after a part of sample contacted and flowed through the bonding pad of two or more detecting systems, makes Bonding pad moves, and thus exposes test film for analysis.In some embodiments, test film is in port openings (portal Opening it is exposed for detecting in).In some embodiments, the bonding pad of two or more detecting systems is to pass through Move removable locking component movement.In some embodiments, make removable locking component movement removable including making Dynamic locking component surrounds the center axis rotation of device.In some embodiments, locking component is moved by laterally or vertically It moves on ground.In some embodiments, it moves locking component and simultaneously or sequentially moves two or more detecting systems Bonding pad.In some embodiments, method further comprises releasing the compression of two or more analyte detection systems. Can for example pressure be released or reduce by making removable locking component movement.In some embodiments, locking is moved Component is the mobile member rotation or mobile (for example, rotation) to move by being connected to removable locking component.
In some embodiments, one or more analysis analyte detection membranous systems are pressed before sample contacts channel system Contracting.In some embodiments, one or more analysis analyte detection membranous systems contact one or more analysis analyte detections in sample It is compressed before the bonding pad of membranous system.In some embodiments, respectively analysis analyte detection membranous system is compressed simultaneously.At some In embodiment, each analyte detection membranous system of analyzing independently is compressed.In some embodiments, each point be present in device Analysis analyte detection membranous system is compressed before sample contacts bonding pad.
In some embodiments, method includes making on two or more analysis analyte detection membranous systems by force member The pressure releases of application, wherein the force member vertically or horizontally moves to release the pressure.In some embodiment party In formula, method includes force member is made to be moved to the second position from first position with pressure relief.In some embodiments, add Power component is moved in power actuator exit or is contacted with power actuator exit, the mobile releasing of force member at this time or reduction pressure Power or releasing or reduction are applied to the power of analysis analyte detection membranous system.In some embodiments, force member is partly or complete (drop out) device is exited entirely.
In it can be used for some embodiments with the device of single signal detection multiple analytes, the present invention provides use In the device of detection and analysis object, it includes pressure actuator, pressure relief device, analysis analyte detection membranous system, analysis analyte detections Membranous system recipient and outlet.In some embodiments, analysis analyte detection membranous system recipient (receptacle) is is enough Receive the size and shape of analysis analyte detection membranous system.In some embodiments, recipient is groove.In some embodiments In, recipient is can (but nonessential) box for being removed from device.
In some embodiments, analysis analyte detection membranous system can be enclosed in or contained in cylinder or outside as described herein In shell.Shell can include first and/or second housing component.In some embodiments, wherein analysis analyte detection membranous system It is accommodated in shell or cylinder, recipient, which has, to be enough to receive the size and shape of shell or cylinder.In some embodiments, outside Shell or cylinder include entrance.Entrance can be used for applying sample to analysis analyte detection membranous system.In some embodiments, cylinder or outer Shell includes the second outlet that sample is allowed to flow through and flow out shell and cylinder.It can be as described herein to analyze analyte detection membranous system Any analysis analyte detection membranous system.
In it can be used for some embodiments with the device of single signal detection multiple analytes, device includes pressure Actuator.For example, pressure actuator can be force member described herein.In some embodiments, it is pressure actuated Device is air valve or vacuum valve, applies air pressure to analysis analyte detection membranous system or analyte detection membranous system is taken out very by analysis It is empty.In some embodiments, can pressure actuator be adjusted by pressure relief device or pressure regulator.Pressure release dress It puts or pressure regulator can be such as vacuum release.Release device or adjuster can be used for adjustment to analysis analyte detection The pressure or vacuum that membranous system applies.Pressure or vacuum can be applied to analysis quality testing by the outlet being present in device or pipe Survey membranous system.Outlet can be the identical outlet that is present herein in the cylinder or shell or its can be different Outlet or pipe.Outlet or pipe can be used so that pressure or vacuum will be applied with specificity rather than it made to expand in whole device It dissipates.
In some embodiments, the shell (for example, cylinder) of cladding analyte film detection including upper case and lower part outside Shell.In some embodiments, shell includes multiple films or pad clamper.In some embodiments, shell include one or Multiple films or pad clamper.In some embodiments, shell includes 1,2,3,4 or 5 film or pad clamper.In some implementations In mode, shell includes at least 1,2,3,4 or 5 film or pad clamper.In some embodiments, shell includes entrance. In some embodiments, shell includes outlet.In some embodiments, vacuum actuator either directly or indirectly contacts shell Outlet.
In it can be used for some embodiments with the device of single signal detection multiple analytes, device and this paper institutes Any device of description includes the injector for spraying shell.Injector can be used for promoting containing analysis analyte detection membrane system The removal of the shell of system.In some embodiments, device includes shell separator.Shell separator can be used for promoting shell Separation.In some embodiments, injector is also used as shell separator.
Other than approach described herein, in some embodiments, detect the methods of multiple analytes also include to Include pressure actuator, pressure regulator, analysis analyte detection membranous system, analysis analyte detection membranous system recipient and outlet or this paper Described any other device analyzes the device of analyte detection membranous system using the sample containing multiple analytes.In some realities It applies in mode, sample is contacted with analysis analyte detection membranous system, wherein flows through to samples vertical analysis analyte detection membranous system.At some In embodiment, method includes the existence or non-existence of detection and analysis object.This can be according to by using phase described herein Bridge joint compound that interaction unit, capture agent and detecting signal unit are formed carries out.
In some embodiments of use device to detect multiple analytes, detection includes removing or move and is present in point The enough amounts of the bonding pad in analyte detection membranous system are analysed so that result is as it can be seen that wherein positive findings indicate depositing for multiple analytes .In some embodiments, detection analyzes analyte detection membranous system and further removal or mobile combination including being removed from device Enough amounts are padded so that single signal is visible to the detection of analyte or multiple analytes.In some embodiments, analyte Therefore detection membranous system is contained in shell or cylinder, and, in some embodiments, shell or cylinder are in mobile or removal knot It is removed before closing pad from device.In some embodiments, as described herein before removal or mobile bonding pad, outside Shell is separated into first (for example, top) and second (for example, lower part) shell.In some embodiments, shell is separated into first It can be removed with second housing or move bonding pad so that test film is visible as described herein.In some embodiments, outside Shell manually and/or is mechanically decoupled.In some embodiments, shell (for example, cylinder) is ejected from device.At some In embodiment, shell is ejected by injector from device.In some embodiments, shell is passed through separator point From.In some embodiments, injector also serves as separator.
In some embodiments, method, which is included on analysis analyte detection membranous system, applies pressure or by analyzing analyte detection Membranous system vacuumizes.In some embodiments, method includes discharging or reducing pressure or vacuum.In some embodiments, It discharges by using pressure regulator or reduces pressure or vacuum.In some embodiments of approach described herein, With the sample that contacts of analysis analyte detection membranous system to flow through analyte membranous system by the flow rate that pressure actuator adjusts.One In a little embodiments, entire sample flows through analysis analyte detection membranous system with constant rate of speed.In some embodiments, sample is with can Variable Rate flows through analysis analyte detection membranous system.In some embodiments, variable bit rate is included at least a period of time one The flow rate for dividing sample is 0.For example, the vacuum that can be adjusted pressure applied or be taken out causes sample to stop running through analysis Analyte detection membranous system continues for some time.This is properly termed as " stopping (dwell) ".As remaining place is retouched in document of the present invention It states, it can be by the way that impermeable or micro- permeable membrane (slightly impermeable membrane) be placed on bonding pad and analyte It is stopped between other layers of detection membranous system to realize.However, it is also possible to it is applied to analysis quality testing by adjusting (for example, change) The pressure of membranous system is surveyed to stop to adjust.It can also be smoked by adjusting (for example, change) by analyzing analyte detection membranous system Vacuum stops to adjust.It can use and adjust through any method for the flow rate for analyzing analyte detection membranous system, including (but not It is limited to) by bonding pad and/or the flow rate of test film.
Device herein can also be automation or is used in combination with optical pickup or other kinds of spectrometer. The advantage that system as described herein and device are combined with optical pickup or other kinds of spectrometer is made to be to increase Device and the sensitivity of analysis cause point for being identified as sample necessary to being positive to analyte to be present in sample It is less to analyse object.Whether this larger sensitivity can contain pathogen, people with certain for subsequent use in food is for example, determined whether Disease or the patient's condition or product whether have in other cases using other device and method use presently described method and Undetectable analyte in same time amount spent by device.
Therefore, in it can be used for some embodiments with the device of single signal detection multiple analytes, the present invention It provides and includes sample inlet, analysis analyte detection cylinder recipient, the cylinder receiving of analysis analyte detection for detecting the device of multiple analytes Device entrance, optional bonding pad remover, pressure actuator, spectrometer (for example, optical pickup), display unit (display Unit), signal processing unit.Pressure actuator can be force member, and position is modified to be applied to and device knot with adjustment Close the pressure of analysis analyte detection membranous system used.Pressure actuator can also be via the analyte by being used in combination with device Detection membranous system vacuumizes to adjust pressure.Spectrometer can be the existing any spectrometer that can detect signal.Signal can Think optical signalling.Signal can be selected from such as infrared spectrum, near infrared spectrum, visible spectrum, x-ray spectrum, ultraviolet The signal emitted in the spectrum of line spectrum, gamma-rays, electromagnetic spectrum etc..
Spectrometer can be connected to signal processing unit (for example, computer).Signal processing unit can be obtained to be transmitted to it Signal and signal Analysis with determine sample existence or non-existence.The example of signal processing unit is (but not limited to) electricity Brain.Signal processing unit can be with sequencing to analyze the signal transmitted by spectrometer.Sequencing can carry out formula with signal Analysis, So that it is determined that in sample analyte existence or non-existence.Formula can be based on pre-installing in the memory of signal processing unit Or criterion input by user by device.The type for the information that can be inputted can be the cut-off (cut- of positive or negative signal Off), processing time etc..Signal processing unit can be also used for adjusting to the pressure for analyzing the application of analyte detection membranous system or passing through The vacuum that analysis analyte detection membranous system is taken out.
Signal processing unit can be used for or be programmed to the flowing speed that adjustment sample flow crosses analysis analyte detection membranous system Rate.Can by adjusting the pressure applied to analysis analyte detection membranous system or by analyze the vacuum that analyte detection membranous system taken out come Adjust flow rate.Sample such as described above for approach described herein, being contacted with analysis analyte detection membranous system To flow through analyte membranous system by the flow rate that pressure actuator adjusts.Pressure regulator in turn can be by such as signal Manage unit adjustment.In some embodiments, entire sample flows through analysis analyte detection membranous system with constant rate of speed, and rate is by signal Processing unit adjusts.In some embodiments, sample flows through analysis analyte detection membranous system with variable bit rate, and rate is by signal Manage unit adjustment.In some embodiments, variable bit rate is included in the flow rate of a part of sample at least a period of time It is 0, flow rate can be adjusted by signal processing unit.For example, can by signal processing unit adjust pressure applied or The vacuum taken out causes sample to stop running through analysis analyte detection membranous system, continues for some time.As discussed herein, this can claim For " stop ".For example, the pressure that analysis analyte detection membranous system can be applied to by adjusting (for example, change) stops to adjust, Adjustment can be realized or be controlled by signal processing unit.It can also be by adjusting (for example, change) by analyzing analyte detection membrane system The taken out vacuum of system stops to adjust, and adjustment can be realized or be controlled by signal processing unit.Adjustment can be used to flow through analysis The flow rate of analyte detection membranous system including but not limited to flows through any side of the flow rate of bonding pad and/or test film Method, and method can be adjusted or be realized by signal processing unit.
In it can be used for some embodiments with the device of single signal detection multiple analytes, herein and in the whole text Described device includes analysis analyte detection cylinder recipient align member (positioning member).Detection cylinder recipient is determined Position component can be used for for example will analysis analyte detection membranous system place it is in place in receive sample and/or for be analyzed Sample.In some embodiments, system is located to analyze for spectrometer.It in some embodiments, can be by believing Number processing unit motorization (motorized) and/or control detection cylinder recipient align member.In some embodiments, it examines Cylinder recipient align member is surveyed without motorization, but can such as (but not limited to) allow to change the position of recipient by personal control Lever or the screw rod control put.In some embodiments, signal processing unit is moved by the way that analyte film is made to detect recipient Component moves the movement to control analyte film detection recipient.In some embodiments, analysis analyte detection cylinder recipient is determined Position component is contacted with analysis analyte detection cylinder recipient.
It is described herein in it can be used for some embodiments with the device of single signal detection multiple analytes Device can include waste receptacle.Waste receptacle can be in device internally or externally, but still contacts device.Waste connects Receiver can receive used analysis analyte detection membranous system.In some embodiments, as described herein, analyte detection is analyzed Membranous system is accommodated in shell (for example, cylinder).Shell can be then injected into waste receptacle.Injection can be manual Or automation.In some embodiments, injection is controlled by signal processing unit.In some embodiments, believe Number processing unit control injector, injector will analyze analyte detection membranous system be ejected into from analysis analyte detection membranous system recipient it is useless In object recipient.All devices as described in this article, in some embodiments, device include analysis analyte detection membrane system System, can be included in or can not be included in shell (for example, cylinder).
In it can be used for some embodiments with the device of single signal detection multiple analytes, pressure actuator connects Touch analysis analyte detection membranous system.In some embodiments, pressure actuator connects at the point that pressure actuator is allowed to move In device.In some embodiments, pressure actuator is in the pivotal point that pressure actuator is allowed to be pivoted at single contact point Place's connection.
In some embodiments, apparatus described herein includes display.In some embodiments, display is Electronic console.In some embodiments, signal processing unit receives the input from spectrometer and on the display unit Show information.Display unit can show the presence of various information, including but not limited to one or more analytes, state etc. And/or it is not present.
In some embodiments, the present invention includes the use of the device comprising signal processing unit or dress described herein It puts to detect multiple analytes.In some embodiments, method includes device is made to contact with sample, in sample contact device Analyze analyte detection membranous system.Sample subsequently passes through analysis analyte detection membranous system.In some embodiments, method includes detection point Analyse the existence or non-existence of object.In some embodiments, detection includes optical pickup detection from analyte membranous system Optical signalling is passed to signal processing unit by optical signalling, optical pickup, and signal processing unit analyzes optical signalling with true The existence or non-existence of setting analysis object；Result is shown on the display unit with signal processing unit.Shown result can be It is vision and/or the sense of hearing.The signal analyzed can be selected from infrared spectrum, near infrared spectrum, visible spectrum, x-ray Spectrum, ultraviolet spectrogram, gamma-rays or electromagnetic spectrum spectrum in signal.In some embodiments, signal is near infrared light In spectrum.In some embodiments, method is ejected into including that will analyze analyte detection membranous system in waste receptacle.In some implementations In mode, signal processing unit is computer.
Refer to the attached drawing, in some embodiments, the embodiment of Fig. 1-Figure 36 drawing apparatus, the component of representative device And it can be used for being combined or not being used in combination specific in method and/or with other devices described herein and/or system Disguise the various views put.
These devices described herein are non-limiting and any other device (including other vertical current devices) It is used equally for bridging compound caused by using a variety of labels and capture agent according to approach described herein to examine Survey multiple analytes.
Fig. 8 descriptions can be used for the device with single signal detection multiple analytes, including the first casing component (10), delay Fliud flushing container (15), second housing component (20), the groove (25) for sliding button, sliding button (30), import (35), ring Bind round (40) and test film (45).Fig. 8 describes the test film (45) for including two kinds of capture agents.First (10) and second (20) shell Component can also be referred to as lower part and upper case component.In Fig. 1, sample will be applied and can be made by import (35) It vertically flows to test film (45).In fig. 8, groove (25) allows sliding button to move, and sliding button is being connected to locking Moving locking member during component, and can move bonding pad in some embodiments and change the position of force member.
Fig. 9 descriptions can be used for the device with single signal detection multiple analytes, including the first casing component (10), the Two casing components (20), the groove (25) for sliding button, sliding button (30), import (35), hoop (40), test film (45), bonding pad (50), multiple absorption components (for example, pad) (55), connecting elements (60), locking component (65) and force member (70).Bonding pad (50), test film (45) and the absorption component (55) that Fig. 9 descriptions are arranged essentially parallel to one another.Force member (70) pressure substantially perpendicular to bonding pad will be applied when being contacted with absorption component.As shown in Figure 9, pass through import (35) The sample contacted with device will pass vertically through bonding pad (50) and flow to test film (45).Although being not explicitly shown in Fig. 9, In some embodiments, permeable membrane is also substantially parallel to the first table of bonding pad (50) and test film (45), wherein permeable membrane The surface of face contact bonding pad (50), the surface of the second surface engaged test film (45) of permeable membrane.
Figure 10 describes bonding pad (50), permeable membrane (75), test film (45) and multiple absorption components (55), can be used for Multiple analytes are detected with single signal.Figure 10 descriptions can be used for the component with single signal detection multiple analytes, base It is parallel to each other in sheet.Figure 10 describes the permeable membrane (75) for including opening.This, which is open, can be used to the result of test film Visualization and detection.
Figure 11 description can be used for single signal detection multiple analytes device, including the first casing component (10), Buffer container (15), second housing component (20), sliding button (30), test film (45), bonding pad (50), permeable membrane (75), multiple absorption components (for example, pad) (55), connecting elements (60), locking component (65) and force member (70).Figure 11 is also Describe the force member (70) comprising axis (72) and head (71), wherein head (71) is than axis (72) width.
Figure 12 descriptions can be used for the partial view of the device with single signal detection multiple analytes, including the first shell Component (10), locking component (65), sliding button (30) and force member (70).Figure 12 descriptions are contacted with force member (70) Locking component (65) causes force member (70) in the method for raising.Figure 12 also describes locking component (65) and sliding button (30) movement far from force member (70) allows force member to change position.In some embodiments, the change of position is Force member reduces.
Figure 13 descriptions can be used for the side sectional view of the device with single signal detection multiple analytes, outside first Mould component (10), second housing component (20), sliding button (30), locking component (65), hoop (40), o-ring (41), reinforcing Component (70) and the supporter (73) for force member.Supporter for axis can be such as the first casing component (10) A part and differentially draw shade merely for example purpose.The button that Figure 13 descriptions are contacted with locking component (65) (30), the way of contact causes the movement of button (30) by moving locking member (65).The mobile of locking component (65) will be from adding Power component (70) takes away supporter, this will allow force member (70) to change position.Figure 13 also describes the axis (72) of force member With head (71).Head (71) generates lip, and wherein locking component (65) can be slided and be supported below force member (70) Force member (70).
Figure 14 descriptions can be used for the partial view of the device with single signal detection multiple analytes, including the first shell Component (10), import (35), test film (45), bonding pad (50), multiple absorption components (55), connects second housing component (20) Connection member (60), locking component (65) and force member (70).Fig. 8 descriptions are connected to bonding pad (50) and locking component (65) Connecting elements (60).Figure 14 also describes the compressed bonding pad of circumference against second housing component (20) and import (35). Figure 14 describes applies the head of stressed force member (71) by contacting multiple absorption components (55).In fig. 14, sample can Device is applied to by import (35) so that sample contacts bonding pad (50) and due to pressure, and sample is contacted by vertical current Test film (45).
Figure 15 A descriptions can be used for the partial view of the device with single signal detection multiple analytes, outside first Mould component (10), second housing component (20), import (35), test film (45), bonding pad (50), multiple absorption components (55), Connecting elements (60), locking component (65) and force member (70).Fig. 8 describes the movement of locking component (65), locking component (65) it is connected to connecting elements (60).Being connected to the movement of the connecting elements (60) of bonding pad (50) moves bonding pad.Figure 15 A Describe the test force member (70) for changing position and the pressure of test film (45) and/or being mitigated or eliminated for compression.Figure 15 C and Figure 15 D also describe movement of the bonding pad (50) far from import (35), appear test film (45) for visualizing and/or detecting.
Figure 16 describes the connecting elements (60) for being connected to bonding pad (50).Figure 16 describes in bonding pad (50) as connection structure The recess (51) for the position that part (60) is connected.Connecting elements can also by other means such as by sticker, bail and its He connects type of attachment.
Figure 17 descriptions can be used for the partial view of the device with single signal detection multiple analytes, including second housing Component (20), multiple pads or film (80) (plurality of pad includes test film, permeable membrane and one or more absorption component) and It can keep the holding member (85) of multiple pads or film (80).Figure 10 describes multiple pads when mobile bonding pad and is retained in appropriate position Structure in putting.Any mode or other structures are used equally for making during multiple pads are held in place.
Figure 18 descriptions can be used for the representative device with single signal detection multiple analytes, outer including further including The first casing component (1002) of shell entrance (1006) and second housing component (1004).In some embodiments, the first He Second housing component can be constructed as single unit.Housing entry allows to introduce the sample into the component of enclosure.Shell enters Mouth, which can have, to be enough to dispose the size for the appropriate volume for being added to the solution in device.In some embodiments, by shell The size for the opening that entrance generates is enough to dispose about 0.1 to about 3ml, about 0.1 to about 2.5ml, about 0.5 to about 2.0ml, about 0.1 About 2.0ml is arrived to about 1.0ml, about 0.5 to about 1.5ml, about 0.5 to about 1.0ml and about 1.0.In some embodiments, device Size cause any size (for example, width, depth or height) be respectively less than or equal to about 5.08cm (2.000 inches).One In a little embodiments, the height of device is less than about 0.635cm (0.250 inch), less than about 0.254cm (0.100 inch), small In about 0.191cm (0.075 inch), less than about 0.165cm (0.065 inch), less than about 0.152cm (0.06 inch) or be less than About 0.140cm (0.055 inch).In some embodiments, the height of device is about 0.127cm (0.050 inch).At some In embodiment, the width or depth of device are less than or equal to about 5.08cm (2.000 inches), about 4.83cm (1.900 English It is very little), about 4.699cm (1.850 inches), about 4.572cm (1.800 inches), about 4.445cm (1.750 inches), about 4.191cm (1.650 inches), about 4.064cm (1.600 inches) or about 3.81cm (1.500 inches).In some embodiments, device Highly it is about 0.127cm (0.050 inch), depth is about 4.445cm (1.750 inches), and width is about 3.81cm (1.500 Inch).
In some embodiments, can be used for including multiple components with the device of single signal detection multiple analytes, Component includes one or more of：Removable member, bonding pad, adhesion component, test film, absorption component, force member, branch Support component or any combination thereof.
In some embodiments, can be used for single signal detection multiple analytes device include force member, Removable member, bonding pad, test film, adhesion component and/or absorption component.In some embodiments, device includes analysis Analyte detection membranous system.In some embodiments, analysis analyte detection membranous system includes bonding pad, test film and absorption component. In some embodiments, analysis analyte detection membranous system includes other permeable membrane, but device can not also contain permeable membrane.In some realities It applies in mode, analysis analyte detection membranous system includes in the following order：Bonding pad, adhesion component, test film and absorption component.
Figure 19 descriptions can be used for the exploded view of the inside of the representative device with single signal detection multiple analytes, dress It puts comprising removable member (1005), bonding pad (1050), adhesion component (1010), test film (1030), absorption component (1040) and supporting member (1020), wherein supporting member further include optional supporting member entrance (1025).It can be removed Component and adhesion component can also be respectively comprising optional removable member entrances (1008) and adhesion means inlet (1012).Group Part can be in the device of such as Figure 18.
Figure 20 descriptions can be used for the representative modul of another representative device with single signal detection multiple analytes, Including adhesion component (1010), supporting member (1020), test film (1030) and absorption component (1040).As shown in Figure 20, Sample can flow through adhesion component (1010) and engaged test film (1030).
Figure 21 describes adhesion component (1010), supporting member (1020), test film (1030) and absorption component (1040).Figure 21 description components are substantially parallel to each other.Figure 21 is further depicted as including the supporting member of supporting member entrance (1025) (1020).This entrance can be used for flowing through device with allowing samples vertical.
Figure 22 partly describes bonding pad (1050), test film (1030) and absorption component (1040).Figure 22, which also describes, to be connect Touch and/or be connected to the bonding pad of removable member (1005).Figure 22 also describes just far from can be used for being detected with single signal The device of multiple analytes removes or mobile removable member, it also makes bonding pad far from device removal or movement.Bonding pad Mobile permission test film visualized, this promotion with single signal analyze and test and analyze object, including multiple analytes.
Figure 23 describes the example of force member (for example, fixture).Representative force member can in a variety of shapes, size and Configuration, but apply pressure on component of each component in force member is positioned over or in force member.Each force member may be used also With comprising opening (+), analyte sample is applied thereto.Figure 23 describes with first component (110) and second component (100) The non-limiting examples of force member.
Figure 24 A, Figure 24 B, Figure 24 C and Figure 24 D partly describe comprising first component (110), b) second component (100), The force member of entrance (115) and analysis analyte detection membranous system (120).Figure 24 A and Figure 24 B also partly describe bonding pad (1050).Bonding pad is invisible in Figure 24 C and Figure 24 D.Figure 24 C and Figure 24 D also partly describe as analysis analyte detection film The test film (1030) of a part for system.Figure 24 D also partly describe by colour band visualization with the test of control responses Film (1030).
Figure 25 partly describe comprising can be removed or removable trimmer (200), entrance (210), bonding pad (1050) and The container of the trimmer of bonding pad (1050).The trimmer (255) of bonding pad can be used for removing bonding pad (1050) from device To expose test film.For example, user can extract the trimmer (255) of bonding pad out to remove bonding pad (1050) from container.Not What is estimated is the analysis analyte detection film compressed between first component as described herein (110) and second component (100) System.
Figure 26 partly describes the first external component (310), the second external component (320), moves or adjustment can be removed Piece (330) and bonding pad (1050).Removable or removable trimmer (330) includes entrance, and entrance exposes bonding pad (1050), Sample is allowd to be applied to bonding pad.Figure 26 does not show the first internal component of compression analysis analyte detection membranous system (120) (110) and the second internal component (100).It can be removed or removable trimmer (330) can move or remove in movement or removal Bonding pad (1050), this allows to estimate and analyze test film.
Removable member entrance in removable member allows to introduce the sample on bonding pad.Entrance, which can have, to be enough Disposition is added to the size of the appropriate volume of the solution in device.In some embodiments, the size of entrance is large enough to locate Put about 0.1 to about 3ml, about 0.1 to about 2.5ml, about 0.5 to about 2.0ml, about 0.1 to about 1.0ml, about 0.5 to about 1.5ml, about 0.5 to about 1.0ml and about 1.0 arrives about 2.0ml.Removable member can also be constructed so that a part for removable member is to molten Liquid is permeable (that is, the region limited by removable member entrance) and another region is impermeable.Permeable regions can For use as entrance, because it will allow solution by removable member and to contact bonding pad.Removable member entrance can have There are many any one of shape and size.In some embodiments, the first casing component is used as removable member.At other In embodiment, the first casing component and removable member are stand-alone assembly.First casing component and removable member wherein For in the embodiment of stand-alone assembly, at least part overlapping of housing entry and removable member entrance allow solution into Enter two entrances.
In some embodiments, the first surface of removable member contact bonding pad.Removable member can also connect In bonding pad.Removable member can be connected to bonding pad when removable member is removed from device or it by any mode During position change, bonding pad is also removed or the position of bonding pad also changes.Removable member can be by such as (but not limited to) Sticker is connected to bonding pad.Sticker includes but is not limited to glue, adhesive tape or will allow removable member and bonding pad each other Other substances of connection.
In some embodiments, removable member directly contacts bonding pad or is combined indirectly by another layer of contact Pad.Sample can be applied to bonding pad directly through the opening in removable member in some embodiments.
Describe to Figure 27 part As the vertical view that can be used for the device with single signal detection multiple analytes, including more A port (2036), entrance (2035) and casing component (2010).Figure 27 A also partly describe visible by port (2301) A part for channel system (2300).Figure 27 part Bs drawing apparatus magnification region, be particularly port (2036). In port, multiple capillaries (2301) are also shown.
Figure 28 descriptions can be used for the bottom view of the device with single signal detection multiple analytes, and device includes multiple power Actuator exit (2200), casing component (2020) and mobile member (2100).
Figure 29 partly describes the first casing component (2010), second housing component (2020), multiple ports (2036), enters Mouthful (2035), channel system (2300), multiple capillaries (2301), bonding pad (2050), multiple test films (2045) and removable Dynamic locking component (2065).The channel system described in Figure 29 is depicted as being made of 3 branches, and branch's number is equal to and is present in dress The number of analysis analyte detection membranous system in putting.
Figure 30 partly describes second housing component (2020), channel system (2300), multiple capillaries (2301), combines Pad (2050), test film (2045) and absorption component (2055) and removable locking component (2065), flexible connecting member (2060), analysis analyte detection membranous system (2400).
Describe to Figure 31 part As multiple power actuator exits (2200), channel system (2300), multiple capillaries (2301), multiple force members (2070), removable locking component (2065), multiple removable locking component extensions (2068), bonding pad (2050), multiple flexible or non-flexible connecting elements extensions (2066) and tubercle (2067), test film (2045) and absorption component (2055).
Figure 31 part Bs device shown in depiction 24A similar portions, however, removable locking component (2065) Center axis rotation is surrounded, and removable locking component extension (2068) no longer supports force member (2070), and adds Power component is returned or drops in power actuator exit (2200).
Figure 32 partly describes the exploded view that can be used for the device with single signal detection multiple analytes, and device includes Channel system (2300), bonding pad (2050), test film (2045), multiple force members (2070), can rotate it is discribed The movable member (2100) of removable locking component (2065).Figure 32 also partly describes removable locking component extension (2068), multiple flexible or non-flexible connecting elements extensions (2066) and tubercle (2067), go out flexible connecting member (2060) Mouthful (2105), second housing component (2020), multiple power actuator exits (2200) and analyze one of analyte detection membranous system Divide (2047).The region of part (2047) comprising analysis analyte detection membranous system has been amplified and has partly described force member (2070), a part for test film (2045), absorption component (2055) and removable locking component extension (2068).
Figure 33 partly describes shell (2020), capillary channel (2301) and channel system (2300).A part of Figure 33 It has been amplified to describe bonding pad (2050), absorption component (2055) and multiple capillaries (2301).
Figure 34 partly describes the sectional view that can be used for the device with single signal detection multiple analytes, including multiple Port (2036), entrance (2035), removable locking component (2065), the movable member for moving the removable locking component (2100), force member (2700), power actuator exit (2200), multiple absorption components (2055), test film (2045) and can Moving locking member extension (2068).Figure 34 also describes the exploded view of a part for analysis analyte detection membranous system, part packet Containing bonding pad (2050), permeable membrane (2056) and absorption component (2055).
Figure 35 partly describes the non-limit of removable locking component (2065) and removable locking component extension (2068) Property example processed.
Figure 36 partly describes the external view and internal view of the shell comprising multiple ports (2036) and entrance (2035).
Figure 37 partly describes the shell comprising multiple power actuator exits (2200) and movable member outlet (2105) Internal view and external view.
Figure 38 partly describe comprising can surround the analysis cylinder (3100) of analyte detection membranous system, power actuator (3200) and Power release device (3000) and outlet (3400) and the device for analyzing analyte detection membranous system recipient (3300).
Figure 39 is partly depicted in the amplification of the outlet (3400) described in Figure 31, recipient (3300) and cylinder (3100) Figure.
Figure 40 partly describes comprising the first casing component (3110), entrance (3135), bonding pad (3350), second housing The exploded view of the cylinder (3100) of component (3120) and multiple film clampers (3122).
Figure 41 partly describes the device for testing and analyzing object, and device includes entrance (3335), membranous system recipient (3300) and display (3500).
Figure 42 is partly depicted in the device of can be used for of describing in Figure 41 of single signal detection multiple analytes It is internal.Device includes the cylinder (3100), membranous system recipient (3300), the power actuator that include analysis analyte detection membranous system (3200), spectrometer is (for example, optical pickup or photodetector (3600), optional bonding pad remover (3201), optional Waste receptacle (3606), motor and membranous system recipient propeller (3605/3607).
Figure 43 is shown in the device of can be used for of describing in Figure 41 and Figure 42 of single signal detection multiple analytes Inside, device are in the various stages being used together with the same components described in Figure 35.Figure 43 A describe cylinder and are just inserted into recipient In.Figure 43 B describe recipient clamping mobile cylinder for sample administration just below entrance, and Figure 43 C are describing sample just It is analyzed by spectrometer.
Figure 44 descriptions can be used for the exploded view of the device with single signal detection multiple analytes, and device is included outside first Mould component (10), second housing component (20), the groove (25) for sliding button, sliding button (30), import (35), test Film (45), bonding pad (50), film (51) in addition, sticker (52), multiple absorption components (for example, pad) (55), connecting elements (60), locking component (65) and force member (70).Can as described herein and/or it is shown assembling component can be made with manufacturing With the device of vertical current detection and analysis object.
Figure 45 descriptions can be used for the optical cable of the device with single signal detection multiple analytes, outside first Mould component (10), the groove (25) for sliding button, sliding button (30), import (35), has no second housing component (20) Test film, bonding pad (50), multiple absorption components (for example, pad) (not shown), connecting elements (60), locking component (do not show Show) and force member (not shown).It can also be manufactured according to approach described herein and be changed using other of this device Form.
Embodiment is described referring now to following embodiment.These embodiments have been merely illustration purpose and provide and embodiment party Formula and should not be construed as being limited to these embodiments, but should be regarded as covering due to provided herein is introduction and become apparent any With all changes form.Those skilled in the art should readily recognize a variety of nonessential parameters, parameter can be varied or It changes to generate substantially similar result.
Embodiment
Embodiment 1：
Two independent PCR reactions are carried out by the use of shiga toxin gene as template, reaction generates the expansion by following label Increase son：1) digoxin and biotin and 2) FITC and biotin.Amplicon then exist in Streptavidin (bridge-jointing unit) or In the absence of mix or respectively enter quick circulation analysis in：Sample A) only amplicon 1, sample B) only amplicon 2 or sample C) with and without amplicon 1+ amplicons 2 under Streptavidin.Circulation analysis is by being coated with anti-digoxin The solid support (nitrocellulose membrane) of (the first capture agent) and the colloidal gold particle composition for being coated with anti-FITC antibody. Under this background, sample C only with Streptavidin generates single positive test signal, and sample A and sample B or does not have The sample C of Streptavidin generates negative test.
Embodiment 2：Multiple analytes are detected using amplicon bridge joint.
Material：
PCR reagent：OneTaq Hot Start polymerases (New England's biology laboratory (New England Biolabs))；5X standard reaction buffer solutions；Haptenization MHALT1.RV (integrated DNA technique company (Integrated DNA Technologies)(IDT))；Haptenization MgC.CH1AS (IDT)；INV018.7E4 VHGene template (ZG)；dNTP； dH2O。
PCR is carried out with the even variable Rate of 3-4 DEG C/s in normal temperature circulator.It is anti-that operation PCR is analyzed by vertical current Should, analysis for example described herein is analyzed, including Veriflow Cassette (stealthy sentry company (Invisible Sentinel))。
Amplicon is generated according to standard scheme.It generates with fluorescein isothiocyanate (FITC) and tetramethylrhodamin (TAMRA) a kind of amplicon of double labeling, and generate and expanded with TAMRA and the another of digoxin (DIG) double labeling Son.DNA cloning reacted from PCR can optionally be precipitated.If precipitated, then can by EtOH or isopropanol+ 1/10v sodium acetates 3M (pH5.2) precipitates to complete.In order to promote to precipitate, 1uL tRNA glycogens can also be added.Make to be deposited in- It carries out continuing minimum 2 hours at 20 DEG C or be carried out at -80 DEG C lasting 15 minutes.The DNA of precipitation is centrifuged about under maximum speed 15 minutes.Liquid is discarded supernatant, DNA precipitations is made to be air-dried 15 minutes.It can be carried out with 70%EtOH ice-cold 20uL optionally Secondary flushing, then centrifugation and drying.Suspension DNA is precipitated with TE (Tris-HCl/EDTA) and makes DNA water again at room temperature About 24 hours.Generated amplicon for universal sequence and does not have specificity to any specific bacteria.
Amplicon is with identifying that the biotinylated antibody of FITC and the antibody of identification rhodamine (that is, TAMRA is marked) mix. Can be with mixtures incubated longer period, such as 5,10,15,20,25 or 30 minutes, but the long period is not required.It is incubated The mixture educated can be added in Veriflow Cassette (vertical current device), and it includes high containing unlabelled anti-ground The test film of pungent antibody and the bonding pad containing Streptavidin-gold conjugate.The presence of device bridge detection compound, bridge joint Compound contains two kinds of amplicons with single signal (colloidal gold).Suitably compare and when generation bridge joint compound must Need all components it is equal in the presence of only detect colloidal gold.It is not intended to be bound to any particular theory, Fig. 3 explanations can use difference The compound that component is formed.When formation bridge joint compound (referring to Fig. 3), colloidal gold signal is detected.Other can also be used The detectable signal of type.If one kind in amplicon is not present, then can't detect signal.Enter it in device in sample Afterwards, it discharges Streptavidin-colloidal gold composite from bonding pad and removes bonding pad.How to generate and using vertical current device Embodiment can be found that in this article with United States Patent (USP) US8,012,770, US 8,183,059 and U.S. patent application case US 13/500,997, in US 13/360,528, US 13/445,233, each case is incorporated by herein by reference.These As a result, it was confirmed that it can specifically detect two kinds of analytes with single detectable signal (being in this embodiment colloidal gold). The detection of signal is not dependent on the precipitation of amplicon after progress PCR reaction steps.
Every patent, patent application case, the disclosure of publication and accession number cited herein is with the side of reference Formula is incorporated by herein.
Although the present invention, which has referred to particular implementation, is subject to disclosure, but it will be apparent that other implementations of the present invention Mode and version can be by those skilled in the art in the case of the true spirit and scope for not departing from the present invention To design.Following claims are intended to be interpreted as including all embodiments and equivalent variations.

Claims (51)

1. a kind of concurrently detect the first analyte of interest and of interest in the test sample for non-diagnostic purpose The method of second analyte, including：
Make solid support and the test sample comprising the first analyte of interest and the second analyte of interest, packet Bridge-jointing unit containing the second capture agent and the detecting signal unit contact comprising third capture agent, wherein, the test specimens First analyte of interest in product is different with second analyte of interest；With
The existence or non-existence of the signal of the detecting signal unit, the letter of the detecting signal unit are detected using single signal Number concurrently indicate the presence of first analyte of interest and the second analyte of interest in the test sample Or be not present, wherein, only when in the test sample first analyte of interest, in the test sample described in Second analyte of interest and the bridge-jointing unit form bridge joint compound, and the detecting signal unit be incorporated into it is described When bridging compound, the single signal is just detected,
Wherein：
First capture agent is fixed on the solid support；
First analyte of interest in the test sample includes the first phase for being incorporated into first capture agent Interaction unit and the second interaction unit for being incorporated into the bridge-jointing unit；And
Second analyte of interest in the test sample includes the first interaction unit and the second interaction Unit, wherein, the first interaction unit of second analyte of interest is with reference to the bridge-jointing unit；
Wherein, the first and second interactions unit of first analyte of interest in the test sample, And the first and second interactions unit of second analyte of interest in the test sample, respectively solely On the spot for heterologous interaction unit, non-natural all mutual of the heterologous interaction unit for corresponding analyte Action cell；
Wherein, the detecting signal unit is incorporated into：I) second analyte of interest in the test sample, ii) The component or iii of the bridge joint compound) only analyzed when the bridge joint compound contains described first in the test sample The component of existing bridge joint compound when object and second analyte.
2. according to the method described in claim 1, wherein, the detecting signal unit is incorporated into the first of second analyte Interact unit or the second interaction unit.
3. according to the method described in claim 1, wherein, the institute of first analyte of interest in the test sample State first interaction of second analyte of interest in the second interaction unit and the test sample Unit includes identical heterologous interaction unit.
4. according to the method described in claim 1, wherein, the institute of first analyte of interest in the test sample State first interaction of second analyte of interest in the second interaction unit and the test sample Unit includes different heterologous interaction units.
5. according to the method described in claim 1, wherein, the institute of first analyte of interest in the test sample State second interaction of second analyte of interest in the first interaction unit and the test sample Unit includes identical heterologous interaction unit.
6. the according to the method described in claim 1, institute of first analyte of interest in wherein described test sample State second interaction of second analyte of interest in the first interaction unit and the test sample Unit includes different heterologous interaction units.
7. according to the method described in claim 1, wherein, first analyte of interest and institute in the test sample Second analyte of interest stated in test sample independently is peptide, sugar, antigen, nucleic acid molecules or their any group It closes.
8. according to the method described in claim 7, wherein, the nucleic acid molecules are amplified production.
9. according to the method described in claim 1, wherein, first analyte of interest and institute in the test sample The second analyte of interest is stated as amplified production.
10. according to the method described in claim 1, wherein, described the first of first analyte of interest interacts Unit is haptens and first capture agent is molecule with reference to the haptens.
11. according to the method described in claim 1, wherein, first analyte of interest in the test sample The first phase interaction of the second interaction unit and second analyte of interest in the test sample With unit it is biotin and second capture agent is compound with reference to biotin.
12. according to the method for claim 11, wherein, second capture agent is Streptavidin.
13. according to the method described in claim 1, wherein, described the second of second analyte of interest interacts Unit is haptens and the third capture agent is molecule with reference to the haptens.
14. according to the method for claim 13, wherein, the third capture agent is to be incorporated into resisting for the haptens Body.
15. according to the method described in claim 1, wherein, the detecting signal unit includes radioactive labels, colloidal gold, glimmering Optical label, nano-particle, quantum dot, magnetic particle or enzyme.
16. according to the method for claim 15, wherein, the nano-particle is radioactive nano particle.
17. a kind of concurrently detect the first analysis of interest with single signal in the test sample for non-diagnostic purpose The method of object, the second analyte of interest and third analyte of interest, including：
Make comprising first analyte of interest, second analyte of interest and the third of interest The test sample of analyte is contacted with solid support, the first bridge-jointing unit, the second bridge-jointing unit and detecting signal unit； With
The presence of the signal of the detecting signal unit is detected, the signal single signal of the detecting signal unit concurrently refers to Show the presence of first analyte of interest, second analyte of interest and the third analyte of interest, In, only when first analyte of interest, second analyte of interest, the third analyte of interest, First bridge-jointing unit and second bridge-jointing unit form bridge joint compound, and the detecting signal unit is incorporated into institute When stating bridge joint compound, the single signal is just detected,
Wherein：
First analyte of interest includes the first interaction unit and the second interaction unit；
Second analyte of interest includes the first interaction unit and the second interaction unit；
The third analyte of interest includes the first interaction unit and the second interaction unit；
The solid support includes the of the first interaction unit for being incorporated into first analyte of interest One capture agent；
First bridge-jointing unit is incorporated into the second interaction unit and described of first analyte of interest The first interaction unit of second analyte of interest；
Second bridge-jointing unit is incorporated into the second interaction unit and described of second analyte of interest The first interaction unit of third analyte of interest；And
The detecting signal unit is incorporated into the second interaction unit of the third analyte of interest；
Wherein, the first and second interactions unit of first analyte of interest, described of interest second The first and second interaction unit of analyte and described the first and second of the third analyte of interest Interaction unit is each independently heterologous interaction unit, and the heterologous interaction unit is corresponding analyte All interaction unit of non-natural.
18. according to the method for claim 17, wherein, first bridge-jointing unit is multivalence capture agent.
19. according to the method for claim 18, wherein, the multivalence capture agent is immunoglobulin.
20. according to the method for claim 19, wherein, the immunoglobulin is IgM.
21. according to the method for claim 17, wherein, second bridge-jointing unit is incorporated into biotin.
22. according to the method for claim 17, wherein, described the first of first analyte of interest interacts Unit is haptens.
23. according to the method for claim 17, wherein, described the second of first analyte of interest interacts Unit is peptide.
24. according to the method for claim 17, wherein, described the first of second analyte of interest interacts Unit is peptide.
25. according to the method for claim 17, wherein, described the second of second analyte of interest interacts Unit is biotin.
26. according to the method for claim 17, wherein, described the first of the third analyte of interest interacts Unit is biotin.
27. according to the method for claim 20, wherein, described the second of the third analyte of interest interacts Unit is haptens.
28. a kind of compound includes the first analyte of interest and of interest second in solid support, test sample Analyte, bridge-jointing unit and detecting signal unit, wherein,
First analyte of interest includes the first interaction unit and the second interaction unit；
Second analyte of interest includes the first interaction unit and the second interaction unit；
Wherein, the first and second interactions unit of first analyte of interest in the test sample, And the first and second interactions unit of second analyte of interest in the test sample, respectively solely On the spot for heterologous interaction unit, non-natural all mutual of the heterologous interaction unit for corresponding analyte Action cell；
Described second point of interest in first analyte of interest, the test sample in the test sample Analyse object, the bridge-jointing unit forms bridge joint compound, and the detecting signal unit is incorporated into the bridge joint compound.
29. compound according to claim 28, wherein：
The solid support includes the first capture agent,
First analyte of interest includes the first interaction unit and the second interaction unit,
Second analyte of interest includes the first interaction unit and the second interaction unit,
The bridge-jointing unit includes one or more second phases for being independently incorporated into first analyte of interest The capture agent of interaction unit and the first interaction unit of second analyte of interest；And
The detecting signal unit includes the second interaction unit for being incorporated into second analyte of interest Capture agent.
30. a kind of compound includes the first analyte of interest, the test sample in solid support, test sample In the second analyte of interest, the third analyte of interest in the test sample, the first bridge-jointing unit, the second bridge Order member and detecting signal unit, wherein, first analyte of interest, the test sample in the test sample In second analyte of interest, the third analyte of interest in the test sample, first bridge Order member, second bridge-jointing unit form bridge joint compound, and to be incorporated into the bridge joint compound for the detecting signal unit Object；Wherein：
First analyte of interest includes the first interaction unit and the second interaction unit；
Second analyte of interest includes the first interaction unit and the second interaction unit；
The third analyte of interest includes the first interaction unit and the second interaction unit；
Wherein, the first and second interactions unit of first analyte of interest, described of interest second The first and second interaction unit of analyte and described the first and second of the third analyte of interest Interaction unit is each independently heterologous interaction unit, and the heterologous interaction unit is corresponding analyte All interaction unit of non-natural.
31. compound according to claim 30, wherein：
The solid support is incorporated into first analyte；
First bridge-jointing unit is incorporated into first analyte of interest and second analyte of interest；
Second bridge-jointing unit is incorporated into second analyte of interest and the third analyte of interest；And
The detecting signal unit is incorporated into the third analyte of interest.
32. compound according to claim 30, wherein
The solid support includes the first capture agent；
First bridge-jointing unit, which includes, one or more is independently incorporated into described the of first analyte of interest The capture agent of two interaction units and the first interaction unit of second analyte of interest；
Second bridge-jointing unit, which includes, one or more is independently incorporated into described the of second analyte of interest The capture agent of two interaction units and the first interaction unit of the third analyte of interest；And
The detecting signal unit includes the second interaction unit for being incorporated into the third analyte of interest Capture agent.
33. a kind of method that multiple analytes are concurrently detected with single signal for non-diagnostic purpose, the method includes：
I) device for detecting multiple analytes in the test sample with single signal is used in one or more comprising a variety of points The sample contact of object is analysed,
Wherein described device includes：
Shell, comprising：
It is in the import that fluid contacts with bonding pad；
Force member；
Contact the slidably locking component of the force member；
Contact the connecting elements of the force member；
Contact the sliding button of the connecting elements；
With the detection membranous system comprising the bonding pad, test film and absorption component,
At least part of the bonding pad, test film and absorption component is parallel to each other,
The force member contact is described to be detected membranous system and can apply the pressure perpendicular to the detection membranous system,
Slidably locking component described in sliding button movement,
The bonding pad includes the detecting signal unit for including third capture agent；
The test film includes the first capture agent for being fixed on the test film；
Wherein one or more of samples include the first analyte of interest, the second analyte of interest and include second The bridge-jointing unit of capture agent,
Wherein described first analyte of interest include be incorporated into first capture agent first interaction unit and It is incorporated into the second interaction unit of the bridge-jointing unit；And second analyte of interest is described comprising being incorporated into First interaction unit of bridge-jointing unit and the second interaction unit；
The wherein described detecting signal unit comprising the third capture agent is incorporated into second analyte, first point described Analyse the component of object and second analyte complex or only when the compound contains first analyte and described second The component of existing bridge-jointing unit during analyte；With
Ii the existence or non-existence of the signal of the detecting signal unit, the detecting signal unit) are detected using single signal Signal parallel indicate the existence or non-existence of first analyte of interest and second analyte of interest, Wherein, it is only bridged when first analyte of interest, second analyte of interest and the bridge-jointing unit are formed Compound, and the detecting signal unit be incorporated into it is described bridge joint compound when, the single signal is just detected.
34. the method according to claim 11, wherein, the detecting signal unit knot for including the third capture agent The first interaction unit or the second interaction unit together in second analyte.
35. according to the method for claim 33, wherein, detection has been included in a part for one or more of samples Contacting and flowing through moves after the bonding pad bonding pad, thus make at least part exposure of the test film with For detecting the detecting signal unit, so as to indicate the existence or non-existence of the multiple analytes with single signal.
36. according to the method for claim 33, wherein, the bonding pad is by moving the slidably locking component Come what is moved.
37. according to the method for claim 33, wherein, one or more of samples are to compress the detection membranous system It is contacted before with the bonding pad.
38. according to the method for claim 33, wherein, first analyte and second analyte are amplicon.
39. according to the method for claim 33, wherein, first analyte and second analyte are reacted for PCR Product.
40. according to the method for claim 33, wherein, the first interaction unit of first analyte is digoxin Label.
41. according to the method for claim 33, wherein, the second interaction unit of first analyte is rhodamine Label.
42. according to the method for claim 33, wherein, the first interaction unit of second analyte is rhodamine Label.
43. according to the method for claim 33, wherein, the second interaction unit of second analyte is fluorescein Label.
44. according to the method for claim 33, wherein, the third capture agent is incorporated into the of second analyte Two interaction units.
45. according to the method for claim 33, wherein, the third capture agent is biotinylated capture agent.
46. according to the method for claim 33, wherein, the detecting signal unit is coated with Streptavidin.
47. according to the method for claim 33, wherein, the detecting signal unit is the colloid of Streptavidin coating Gold.
48. according to the method for claim 33, wherein, first analyte and second analyte are nucleic acid amplification Product, wherein：
First analyte includes digoxigenin labeled and rhodamine marks；
Second analyte includes rhodamine label and fluorescein label；
First capture agent is anti-digoxigenin labeled antibody；
Second capture agent is anti-rhodamine labelled antibody；
The third capture agent is biotinylated anti-fluorescein labelled antibody；And
The detecting signal unit is the colloidal gold of Streptavidin coating.
49. according to the method described in claim 1, wherein, the test sample is foodstuff samples.
50. according to the method described in claim 1, wherein, first analyte of interest and described of interest second Analyte comes from same organism.
51. according to the method for claim 50, wherein, the organism for Escherichia coli, Listeria, Campylobacter or Salmonella.
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CN104919055A (en) 2015-09-16
US20160340714A1 (en) 2016-11-24
AU2013230917C1 (en) 2021-09-16
US10732177B2 (en) 2020-08-04
US9347938B2 (en) 2016-05-24
EP3578980B1 (en) 2021-09-15
EP2823308A2 (en) 2015-01-14
JP6190395B2 (en) 2017-08-30
HK1205760A1 (en) 2015-12-24
AU2013230917A1 (en) 2014-09-25

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